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Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) is the first reported nonreceptor oncogenic tyrosine phosphatase connecting multiple signal transduction cascades and exerting immunoinhibitory function through the PD-1 checkpoint receptor. As part of a drug discovery program aimed at obtaining novel allosteric SHP2 inhibitors, a series of pyrazopyrazine derivatives bearing an original bicyclo[3.1.0]hexane basic moiety on the left-hand side region of the molecule were identified. We report herein the discovery process, the in vitro pharmacological profile, and the early developability features of compound 25 , one of the most potent members of the series., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

Protein tyrosine phosphatase SHP2 is an oncogenic protein that can regulate different cytokine receptor and receptor tyrosine kinase signaling pathways. We report here the identification of a novel series of SHP2 allosteric inhibitors having an imidazopyrazine 6,5-fused heterocyclic system as the central scaffold that displays good potency in enzymatic and cellular assays. SAR studies led to the identification of compound 8 , a highly potent SHP2 allosteric inhibitor. X-ray studies showed novel stabilizing interactions with respect to known SHP2 inhibitors. Subsequent optimization allowed us to identify analogue 10 , which possesses excellent potency and a promising PK profile in rodents., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

Background: Artificial intelligence (AI) has the potential to enhance patient safety in surgery, and all its aspects, including education and training, will derive considerable benefit from AI. In the present study, deep-learning models were used to predict the rates of proficiency acquisition in robot-assisted surgery (RAS), thereby providing surgical programs directors information on the levels of the innate ability of trainees to facilitate the implementation of flexible personalized training., Methods: 176 medical students, without prior experience with surgical simulators, were trained to reach proficiency in five tasks on a virtual simulator for RAS. Ensemble deep neural networks (DNN) models were developed and compared with other ensemble AI algorithms, i.e., random forests and gradient boosted regression trees (GBRT)., Results: DNN models achieved a higher accuracy than random forests and GBRT in predicting time to proficiency, 0.84 vs. 0.70 and 0.77, respectively (Peg board 2), 0.83 vs. 0.79 and 0.78 (Ring walk 2), 0.81 vs 0.81 and 0.80 (Match board 1), 0.79 vs. 0.75 and 0.71 (Ring and rail 2), and 0.87 vs. 0.86 and 0.84 (Thread the rings 2). Ensemble DNN models outperformed random forests and GBRT in predicting number of attempts to proficiency, with an accuracy of 0.87 vs. 0.86 and 0.83, respectively (Peg board 2), 0.89 vs. 0.88 and 0.89 (Ring walk 2), 0.91 vs. 0.89 and 0.89 (Match board 1), 0.89 vs. 0.87 and 0.83 (Ring and rail 2), and 0.96 vs. 0.94 and 0.94 (Thread the rings 2)., Conclusions: Ensemble DNN models can identify at an early stage the acquisition rates of surgical technical proficiency of trainees and identify those struggling to reach the required expected proficiency level., (© 2022. The Author(s).)

Hypothesis: Calciprotein particles (CPPs) are endogenous nanoparticles consisting of hybrid mineral-organic colloidal complexes made of calcium phosphates and Fetuin-A (Fet-A), a protein that in physiological conditions binds to amorphous calcium phosphate forming primary CPP (CPP1). CPP1 can crystallize resulting in hydroxyapatite-based secondary CPP (CPP2) that can eventually precipitate leading to vascular calcifications. The treatment of patients with molecules and ions that delay the amorphous-to-crystalline transition has shown promising results from a clinical perspective, but the study of their mechanism of action has not been thoroughly examined so far., Experiments: This work describes the formation and crystallization mechanism of synthetic analogs of endogenous CPPs. The effect of different concentrations of Fet-A and of stabilizing agents (Mg 2+ , citrate and pyrophosphate) on the features and stability of CPPs was addressed by combining different characterization techniques such as turbidimetry, dynamic light scattering, infrared spectroscopy, and scanning electron microscopy., Findings: The results show that the stabilizing agents display different action mechanisms and are effective to a different extent in preventing the formation of CPPs or delaying their crystallization. Such findings could be of interest to develop effective therapies for vascular calcifications and to deepen the understanding of amorphous calcium phosphate stabilization and its interaction with proteins., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: This study aimed at standardizing and providing preliminary evidence on the clinical usability of the Italian telephone-based Verbal Fluency Battery (t-VFB), which includes phonemic (t-PVF), semantic (t-SVF) and alternate (t-AVF) verbal fluency tasks., Methods: Three-hundred and thirty-five Italian healthy participants (HPs; 140 males; age range = 18-96 years; education range = 4-23 years) and 27 individuals with neurodegenerative or cerebrovascular diseases were administered the t-VFB. Switch number and cluster size were computed via latent semantic analyses. HPs underwent the telephone-based Mental State Examination (MMSE) and Backward Digit Span (BDS). Construct validity, factorial structure, internal consistency, test-retest and inter-rater reliability and equivalence with the in-person Verbal Fluency tasks were assessed. Norms were derived via Equivalent Scores. Diagnostic accuracy against clinical populations was assessed., Results: The majority of t-VFB scores correlated among each other and with the BDS, but not with the MMSE. Switch number correlated with t-PVF, t-SVF, t-AVF scores, whilst cluster size with the t-SVF and t-AVF scores only. The t-VFB was underpinned by a mono-component structure and was internally consistent (Cronbach's α = 0.91). Test-retest (ICC = 0.69-0.95) and inter-rater reliability (ICC = 0.98-1) were optimal. Each t-VFB test was statistically equivalent to its in-person version (equivalence bounds yielding a p < 0.05). Education predicted all t-VFB scores, whereas age t-SVF and t-AVF scores and sex only some t-SVF scores. Diagnostic accuracy against clinical samples was optimal (AUC = 0.81-0.86)., Discussion: The t-VFB is a valid, reliable and normed telephone-based assessment tool for language and executive functioning, equivalent to the in-person version; results show promising evidence of its diagnostic accuracy in neurological populations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Aiello, Preti, Pucci, Diana, Corvaglia, Barattieri di San Pietro, Difonzo, Zago, Appollonio, Mondini and Bolognini.)

Background: The aim of this paper is to present a freely accessible new instrument for the evaluation of cognition: the Global Examination of Mental State (GEMS)., Methods: It is made up of 11 items tapping into a range of skills, such as Orientation in time and space, Memory, Working memory, Visuo-spatial, Visuo-constructional and Planning abilities, Perceptual and visual Attention, Language (Naming, Comprehension, and Verbal fluency), and Pragmatics., Results: The psychometric strengths of this screening are: (1) extensive and updated normative data on the adult Italian population (from 18 to 100 years old); (2) absence of ceiling effect in healthy individuals, which allows to better detect interindividual variability; (3) comparison of the global scores with normative data taking into account Cognitive Reserve rather than only education, thus increasing diagnostic accuracy; (4) thresholds for significant change over time and the possibility to use parallel versions (GEMS-A/GEMS-B) for test-retest; (5) solid psychometric properties and data on discriminant validity; and (6) free access to all materials (record forms, instructions, and cut-off scores) on the web under a Creative Common License., Conclusions: With all these characteristics, GEMS could be a very useful paper-and-pencil instrument for cognitive screening., (© 2022 The Authors. Brain and Behavior published by Wiley Periodicals LLC.)

Background: Conventional Right Colectomy with D2 lymphadenectomy (RC-D2) currently represent the most common surgical treatment of right-sided colon cancer (RCC). However, whether it should be still considered a standard of care, or replaced by a routine more extended D3 lymphadenectomy remains unclear. In the present study, we aim to critically review the patterns of relapse and the survival outcomes obtained from our 11-year experience of RC-D2., Methods: Clinical data of 489 patients who underwent RC-D2 for RCC at two centres, from January 2009 to January 2020, were retrospectively reviewed. Patients with synchronous distant metastases and/or widespread nodal involvement at diagnosis were excluded. Post-operative clinical-pathological characteristics and survival outcomes were evaluated including the pattern of disease relapse., Results: We enrolled a total of 400 patients with information follow-up. Postoperative morbidity was 14%. The median follow-up was 62 months. Cancer recurrence was observed in 55 patients (13.8%). Among them, 40 patients (72.7%) developed systemic metastases, and lymph-node involvement was found in 7 cases (12.8%). None developed isolated central lymph-node metastasis (CLM), in the D3 site. The estimated 3- and 5-year relapse-free survival were 86.1% and 84.4%, respectively. The estimated 3- and 5-year cancer-specific OS were 94.5% and 92.2%, respectively., Conclusions: The absence of isolated CLM, as well as the cancer-specific OS reported in our series, support the routine use of RC-D2 for RCC. However, D3 lymphadenectomy may be recommended in selected patients, such as those with pre-operatively known CLM, or with lymph-node metastases close to the origin of the ileocolic vessels., (© 2022. The Author(s).)

Many peptide drugs such as insulin and glucagon-like peptide (GLP-1) analogues are successfully administered subcutaneously (SC). Following SC injection, peptides may undergo catabolism in the SC compartment before entering systemic circulation, which could compromise their bioavailability and in turn affect their efficacy.This review will discuss how both technology and strategy have evolved over the past years to further elucidate peptide SC catabolism.Modern bioanalytical technologies (particularly liquid chromatography-high-resolution mass spectrometry) and bioinformatics platforms for data mining has prompted the development of in silico , in vitro and in vivo tools for characterising peptide SC catabolism to rapidly address proteolytic liabilities and, ultimately, guide the design of peptides with improved SC bioavailability.More predictive models able to recapitulate the interplay between SC catabolism and other factors driving SC absorption are highly desirable to improve in vitro/in vivo correlations.We envision the routine incorporation of in vitro and in vivo SC catabolism studies in ADME screening funnels to develop more effective peptide drugs for SC delivery.

Background: Despite the relevance of telephone-based cognitive screening tests in clinical practice and research, no specific test assessing executive functioning is available. The present study aimed at standardizing and providing evidence of clinical usability for the Italian telephone-based Frontal Assessment Battery (t-FAB)., Methods: The t-FAB (ranging 0-12), comprising two subtests, has two versions: one requiring motor responses (t-FAB-M) and the other verbal responses (t-FAB-V). Three hundred and forty-six Italian healthy adults (HPs; 143 males; age range = 18-96 years; education range = 4-23 years) and 40 participants with neurological diseases were recruited. To HPs, the t-FAB was administered along with a set of telephone-based tests: MMSE, verbal fluency (VF), backward digit span (BDS). The in-person version of the FAB was administered to both HPs and clinical groups. Factorial structure, construct validity, inter-rater and test-retest reliability, t-FAB-M vs. t-FAB-V equivalence and diagnostic accuracy were assessed. Norms were derived via Equivalent Scores., Results: In HPs, t-FAB measures yielded high inter-rater/test-retest reliability (ICC = .78-.94), were internally related (p ≤ .005) and underpinned by a single component, converging with the telephone-based MMSE, VF, BDS (p ≤ .0013). The two t-FAB versions were statistically equivalent in clinical groups (ps of both equivalence bounds < .001). Education predicted all t-FAB scores (p < .001), whereas age only the t-FAB-M score (p ≤ .004). t-FAB scores converge with the in-person FAB in HPs and clinical groups (r s  = .43-.78). Both t-FAB versions were accurate in discriminating HPs from the clinical cohort (AUC = .73-.76)., Discussion: The t-FAB is a normed, valid, reliable and clinically usable telephone-based cognitive screening test to adopt in both clinical and research practice., (© 2022. The Author(s).)

Background: The Italian telephone-based Mini-Mental State Examination (Itel-MMSE), despite being psychometrically sound, has shown relevant ceiling effects, which may negatively impact the interpretation of its scores. In address to overcome such an issue, this study aimed at providing item-level insights on the Itel-MMSE through Item Response Theory (IRT) analyses., Methods: Five-hundred and sixty-seven healthy Italian adults (227 males, 340 females; mean age: 51 ± 17 years, range 18-96; mean education: 13.31 ± 4.3 years). A two-parameter logistic IRT model was implemented to assess item discrimination and difficulty of the Itel-MMSE. Construct unidimensionality, statistical independence of items, and model and item fit were tested. Informativity levels were also assessed graphically., Results: With respect to the Itel-MMSE total score, ceiling effects were found in 92.7% of participants. Unidimensionality was violated; both model and item fit were poor; a few items showed statistical dependence. Both the whole test and its items proved to be scarcely informative, especially for medium-to-high levels of ability, except for attention and spatial orientation subtests, which consistently yielded the highest discriminative capability., Discussion: The Itel-MMSE appears to be most informative in low-performing healthy individuals. However, the present findings should not lead practitioners to aprioristically equate ceiling effects/low informativity to clinical uselessness. Items assessing attention and, to a lesser extent, spatial orientation appear to be the most informative., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background and Purpose: Many different factors have been hypothesized to modulate cognition in an aging population according to their functioning at baseline., Methods: This retrospective study quantifies the relative contribution of age and sex as demographic factors, comorbidity, education and occupation (classified with the International Standard Classification of Occupation 2008) as cognitive reserve proxies in accounting for cognitive aging. All participants (3081) were evaluated at baseline with a complete neuropsychological test battery (T1) and those with unimpaired profiles were classified as subjective cognitive decline, those mildly impaired as mild neurocognitive decline and those severely impaired as major neurocognitive decline. From the first assessment 543 individuals were assessed a second time (T2), and 125 a third time (T3). Depending on whether they maintained or worsened their profile, based on their initial performance, participants were then classified as resistant or declining., Results: At baseline, all individuals showed education and occupation as the best predictors of performance, in addition to age. Furthermore, across assessments, the resistant had higher levels of education and occupation than the declining. In particular, the education and occupation predicted cognitive performance in all groups considered, from the subjective cognitive decline to the one with the most severely impaired participants., Conclusions: This study highlights the role of working activity in protecting from cognitive decline across all fragile elderly groups and even more so the individuals who are at very high risk of decline., (© 2021 The Authors. European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

The innate immune agonist STING (STimulator of INterferon Genes) binds its natural ligand 2'3'-cGAMP (cyclic guanosine-adenosine monophosphate) and initiates type I IFN production. This promotes systemic antigen-specific CD8 + T-cell priming that eventually provides potent antitumor activity. To exploit this mechanism, we synthesized a novel STING agonist, MSA-1, that activates both mouse and human STING with higher in vitro potency than cGAMP. Following intratumoral administration of MSA-1 to a panel of syngeneic mouse tumors on immune-competent mice, cytokine upregulation and its exposure were detected in plasma, other tissues, injected tumors, and noninjected tumors. This was accompanied by effective antitumor activity. Mechanistic studies in immune-deficient mice suggested that antitumor activity of intratumorally dosed STING agonists is in part due to necrosis and/or innate immune responses such as TNF-α activity, but development of a robust adaptive antitumor immunity is necessary for complete tumor elimination. Combination with PD-1 blockade in anti-PD-1-resistant murine models showed that MSA-1 may synergize with checkpoint inhibitors but can also provide superior tumor control as a single agent. We show for the first time that potent cyclic dinucleotides can promote a rapid and stronger induction of the same genes eventually regulated by PD-1 blockade. This may have contributed to the relatively early tumor control observed with MSA-1. Taken together, these data strongly support the development of STING agonists as therapy for patients with aggressive tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 treatment by enhancing the anti-PD-1 immune profile., (©2021 American Association for Cancer Research.)

Prostaglandin E 2 (PGE 2 ), an arachidonic acid pathway metabolite produced by cyclooxygenase (COX)-1/2, has been shown to impair anti-tumor immunity through engagement with one or more E-type prostanoid receptors (EP 1-4 ). Specific targeting of EP receptors, as opposed to COX-1/2 inhibition, has been proposed to achieve preferential antagonism of PGE 2 -mediated immune suppression. Here we describe the anti-tumor activity of MF-766, a potent and highly selective small-molecule inhibitor of the EP 4 receptor. EP 4 inhibition by MF-766 synergistically improved the efficacy of anti-programmed cell death protein 1 (PD-1) therapy in CT26 and EMT6 syngeneic tumor mouse models. Multiparameter flow cytometry analysis revealed that treatment with MF-766 promoted the infiltration of CD8 + T cells, natural killer (NK) cells and conventional dendritic cells (cDCs), induced M1-like macrophage reprogramming, and reduced granulocytic myeloid-derived suppressor cells (MDSC) in the tumor microenvironment (TME). In vitro experiments demonstrated that MF-766 restored PGE 2 -mediated inhibition of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production in THP-1 cells and human blood, and PGE 2 -mediated inhibition of interleukin (IL)-2-induced interferon (IFN)-γ production in human NK cells. MF-766 reversed the inhibition of IFN-γ in CD8 + T-cells by PGE 2 and impaired suppression of CD8 + T-cells induced by myeloid-derived suppressor cells (MDSC)/PGE 2. In translational studies using primary human tumors, MF-766 enhanced anti-CD3-stimulated IFN-γ, IL-2, and TNF-α production in primary histoculture and synergized with pembrolizumab in a PGE 2 high TME. Our studies demonstrate that the combination of EP 4 blockade with anti-PD-1 therapy enhances antitumor activity by differentially modulating myeloid cell, NK cell, cDC and T-cell infiltration profiles., (© 2021 Merck and Co. Published with license by Taylor & Francis Group, LLC.)

Drug discovery is moving at a rapid pace and a fast turnaround of bioanalytical data is needed to sustain this pace. This article focuses on the evaluation of time-saving homogeneous proximity immunoassays such as Amplified Luminescent Proximity Homogeneous Assay, Time-Resolved Fluorescence Resonance Energy Transfer and Spatial Proximity Analyte Reagent Capture Luminescence as an alternative to industry popular platforms like mesoscale discovery (MSD) and Gyrolab ® . Our evaluation showed that no one platform can be considered the best for all the parameters assessed. Homogeneous proximity platforms were found to be advantageous over MSD and Gyrolab for certain applications and are herein discussed. The factors affecting the performance of homogeneous assays and appropriate corrections are discussed. The homogeneous assays, due to their flexibility, hold a lot of untapped potential for the future of bioanalysis.

Objective: To investigate whether and when palliative sedation was discussed with hospice patients with cancer and/or with their families and factors associated with patient involvement in such discussions., Methods: Medical records of all patients with cancer who died in an Italian hospice in 2014-2015 (N = 326) were retrospectively reviewed. Multiple logistic regression was used to assess the association between patients' characteristics and palliative sedation discussion with the patient versus palliative sedation discussion only with the family., Results: Palliative sedation discussion was in 51.8% of the cases reported in the record. In most of the cases, discussions were conducted pre-emptively. Palliative sedation was used for 67.3% of the patients who were involved in the discussion and for 32.7% of the patients when the topic was discussed only with the family. Patient involvement in palliative sedation discussions was negatively associated with living with others (OR 0.34, CI 0.15; 0.77), and positively associated with awareness of prognosis (OR 5.61, CI 2.19; 14.33) and days of survival after hospice admission (OR 3.41, CI 1.55; 7.51)., Conclusion: Policies encouraging patient involvement in palliative care decision-making, including palliative sedation, should be implemented and their adoption should be carefully examined. Prospective studies addressing this topic are needed., (© 2019 John Wiley & Sons Ltd.)

We report herein the design and synthesis of a series of orally active, liver-targeted hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) inhibitors for the treatment of anemia. In order to mitigate the concerns for potential systemic side effects, we pursued liver-targeted HIF-PHD inhibitors relying on uptake via organic anion transporting polypeptides (OATPs). Starting from a systemic HIF-PHD inhibitor ( 1 ), medicinal chemistry efforts directed toward reducing permeability and, at the same time, maintaining oral absorption led to the synthesis of an array of structurally diverse hydroxypyridone analogues. Compound 28a was chosen for further profiling, because of its excellent in vitro profile and liver selectivity. This compound significantly increased hemoglobin levels in rats, following chronic QD oral administration, and displayed selectivity over systemic effects., Competing Interests: The authors declare no competing financial interest.

Mutations in IDH1 are highly prevalent in human glioma. First line treatment is radiotherapy, which many patients often forego to avoid treatment-associated morbidities. The high prevalence of IDH1 mutations in glioma highlights the need for brain-penetrant IDH1 mutant-selective inhibitors as an alternative therapeutic option. Here, we have explored the utility of such an inhibitor in IDH1 mutant patient-derived models to assess the potential therapeutic benefits associated with intracranial 2-HG inhibition. Treatment of mutant IDH1 cell line models led to a decrease in intracellular 2-HG levels both in vitro and in vivo. Interestingly, inhibition of 2-HG production had no effect on in vitro IDH1 mutant glioma cell proliferation. In contrast, IDH1 mutant-selective inhibitors provided considerable survival benefit in vivo. However, even with near complete inhibition of intratumoral 2-HG production, not all mutant glioma models responded to treatment. The results suggest that disruption of 2-HG production with brain-penetrant inhibitors in IDH1 mutant gliomas may have substantial patient benefit.

Purpose: To describe the outcomes of intravitreal anti-vascular endothelial growth factor (VEGF) in the treatment of retinal angiomatous proliferation (RAP) in real-life practice in 7 Italian centers under the Progetto Luce initiative., Methods: Clinical data of 95 eyes of 95 patients affected by RAP, regularly followed up and treated with either intravitreal ranibizumab or bevacizumab over 12 months, were examined. After a loading phase of 3 consecutive injections, retreatments were administered following a pro-re-nata regimen on the basis of the persistence or the recurrence of subretinal/intraretinal fluid on optical coherence tomography, or leakage on fluorescein angiography., Results: Overall, the mean best corrected visual acuity changed from 0.66 to 0.53 LogMAR (P: 0.0003); 36.8% of eyes gained at least 3 ETDRS lines, whereas 13.7% lost >3 lines at the end of the follow-up. Mean central retinal thickness improved from 384 μm at baseline to 262 μm at the 12-month examination (P < 0.001). A serous pigment epithelium detachment (PED) was identified in 68.4% of eyes at baseline, and it was still detectable in 30.5% at the end of follow-up. The mean number of injections was 4.4 over the follow-up. A significantly greater proportion of eyes showed PED resolution in the subgroup treated with ranibizumab (P < 0.001)., Conclusions: Intravitreal anti-VEGF treatment in routine clinical practice allows a significant improvement in visual function in patients affected by RAP. A limited number of anti-VEGF injections are generally required in most cases.

Study Objective: We investigated whether rupture increased the recurrence rate of pediatric ovarian neoplasms., Design: 20-year single-institution retrospective study., Setting: Tertiary, free-standing, university children's hospital., Participants: All girls with ovarian neoplasms treated during between 1991 and 2011., Main Outcome Measure: Tumor recurrence., Results: Fifty-nine tumors in 53 patients were managed, including 51/59 (86%) benign and 8/59 (14%) malignant. Laparotomy was employed in 44/59 (75%), laparoscopy in 8/59 (14%), and laparoscopy converted to laparotomy in 7/59 (12%). Total and partial oophorectomy (cystectomy) was used for 15/51 (29%) and 36/51 (71%) of benign tumors, respectively. All malignant tumors underwent total oophorectomy. Accidental rupture or intentional tumor puncture occurred in 26/56 cases (46%), 23/51 benign and 3/5 malignant. Rupture was associated with increasing cyst size on univariate and multivariate analyses (p = 0.002 and p = 0.004, respectively). There were 5 recurrences (9%) in 4 patients, including 4 benign (3 mature teratomas, 1 mucinous cystadenoma), and 1 malignant yolk sac tumor. Recurrence occurred in 2/30 (7%) without rupture and 3/26 (12%) with rupture, p = 0.66. Follow-up was available for 50/53 patients (94%), with a median of 23.8 months [range 0.2-189 months]. All recurrences were salvaged by surgery., Conclusions: In this limited study, intra-operative rupture did not increase the recurrence rate or worsen the prognosis of pediatric ovarian neoplasms., (Copyright © 2016 North American Society for Pediatric and Adolescent Gynecology. Published by Elsevier Inc. All rights reserved.)

Purpose: Wee1 regulates key DNA damage checkpoints, and in this study, the efficacy of the Wee1 inhibitor MK-1775 was evaluated in glioblastoma multiforme (GBM) xenograft models alone and in combination with radiation and/or temozolomide., Experimental Design: In vitro MK-1775 efficacy alone and in combination with temozolomide, and the impact on DNA damage, was analyzed by Western blotting and γH2AX foci formation. In vivo efficacy was evaluated in orthotopic and heterotopic xenografts. Drug distribution was assessed by conventional mass spectrometry (MS) and matrix-assisted laser desorption/ionization (MALDI)-MS imaging., Results: GBM22 (IC50 = 68 nmol/L) was significantly more sensitive to MK-1775 compared with five other GBM xenograft lines, including GBM6 (IC50 >300 nmol/L), and this was associated with a significant difference in pan-nuclear γH2AX staining between treated GBM22 (81% cells positive) and GBM6 (20% cells positive) cells. However, there was no sensitizing effect of MK-1775 when combined with temozolomide in vitro. In an orthotopic GBM22 model, MK-1775 was ineffective when combined with temozolomide, whereas in a flank model of GBM22, MK-1775 exhibited both single-agent and combinatorial activity with temozolomide. Consistent with limited drug delivery into orthotopic tumors, the normal brain to whole blood ratio following a single MK-1775 dose was 5%, and MALDI-MS imaging demonstrated heterogeneous and markedly lower MK-1775 distribution in orthotopic as compared with heterotopic GBM22 tumors., Conclusions: Limited distribution to brain tumors may limit the efficacy of MK-1775 in GBM., (©2015 American Association for Cancer Research.)

In this study, we analyze the management of ovarian masses in a total of 130 children surgically treated for 137 ovarian lesions (7 bilateral). The most frequent symptoms were chronic (52.3%) and acute (25.4%) abdominal pain. Histological examination revealed 64 (46.7%) functional lesions, 59 (43.1%) benign neoplasms, 5 (3.7%) malignant ones, and 7 (6.6%) torsed normal ovaries. Ovarian torsion occurred in 36 cases (26.27%). A conservative treatment was performed in 81 (59.1%) girls: 61 (75.3%) treated in nonemergency and 20 (24.7%) in emergency surgery; laparoscopic approach in 35 cases (43.2%); and open surgery in 46 (56.8%). The remaining 56 (40.9%) ovarian masses underwent nonconservative surgery: 40 cases (71.4%) nonemergency and 16 (28.6%) emergency; laparoscopy in 20 patients (35.7%); and open surgery in 36 (64.3%). Fertility preservation should be a goal in the surgical treatment. The management of ovarian torsion should include adnexal detorsion and recovery of the ovarian tissue. In case of benign neoplasms, laparoscopic tumorectomy should be the gold standard; in early stage malignant tumors, fertility-sparing surgery with accurate staging is preferred.

Dinaciclib is a potent CDK1, 2, 5 and 9 inhibitor being developed for the treatment of cancer. Additional understanding of antitumor mechanisms and identification of predictive biomarkers are important for its clinical development. Here we demonstrate that while dinaciclib can effectively block cell cycle progression, in vitro and in vivo studies, coupled with mouse and human pharmacokinetics, support a model whereby induction of apoptosis is a main mechanism of dinaciclib's antitumor effect and relevant to the clinical duration of exposure. This was further underscored by kinetics of dinaciclib-induced downregulation of the antiapoptotic BCL2 family member MCL1 and correlation of sensitivity with the MCL1-to-BCL-xL mRNA ratio or MCL1 amplification in solid tumor models in vitro and in vivo. This MCL1-dependent apoptotic mechanism was additionally supported by synergy with the BCL2, BCL-xL and BCL-w inhibitor navitoclax (ABT-263). These results provide the rationale for investigating MCL1 and BCL-xL as predictive biomarkers for dinaciclib antitumor response and testing combinations with BCL2 family member inhibitors.

Purpose: The aim of this study was to investigate the effects of gonadotropin releasing hormone analog (GnRHa) therapy on normalized testicular atrophy index (TAIn) using gonadorelin before and after orchiopexy., Methods: 62 infants with 87 undescended testes (UDT) were prospectively assigned to two homogeneous groups according to age, position of UDT and TAIn. The patients were randomized to receive either orchiopexy alone or orchiopexy combined with GnRHa as nasal spray at 1.2 mg daily for 4 weeks before surgery and 4 weeks after surgery. Surgical approaches were relative to the position of the UDT: Shoemakers technique in proximal-UDT and Bianchi technique in distal-UDT. All the patients were evaluated clinically and sonographically 1 month before surgery, at the time of surgery, 1 month, 6 months and 5 years after surgery., Results: Ultrasound data in our study have shown a statistically significant decrease of TAIn in children given additional HT only after 5 years of follow-up, in unilateral cases and in the entirety of treated patients; in bilateral cases this difference was not statistically significant., Conclusion: Patients with a TAIn >20% treated with preoperative and post-operative GnRHa therapy have a significant increase in testicular volume after 5 years of follow-up, as shown by the relative reduction of TAIn values.

Merck consolidated discovery stage bioanalytical functions into the Department of Pharmacokinetics, Pharmacodynamics & Drug Metabolism in 2007. Since then procedures and equipment used to provide important quantitative data to project teams have been harmonized and in many cases standardized. This approach has enabled movement of work across the network of laboratories and has resulted in a lean, flexible and efficient organization. The overall goal was to reduce time and resources spent on routine activities while creating time to perform research in new areas and technologies to support future scientific needs. The current state of discovery bioanalysis at Merck is discussed, including hardware and software platforms, workflow procedures and performance metrics. Examples of improved processes will be discussed for compound tuning, LC method development, analytical acceptance criteria, automated sample preparation, sample analysis platforms, data processing and data reporting.

This paper communicates Merck's thoughts on why, when and how to use dried blood spot (DBS) technology in a clinical setting, and provides a strategic approach, emphasizing the necessary steps, for successful clinical implementation of this microsampling technique. PK consideration based on relevant in vitro data, that is, blood-to-plasma ratio, hematocrit, plasma unbound fraction and/or blood cell partition, is suggested to be part of the decision tree on when to choose DBS as a surrogate matrix for PK analysis. A quick feasibility assessment addressing analytical challenges, including sensitivity, hematocrit impact and storage stability, needs to be evaluated before initiating DBS studies. Special attention should be paid to the clinical sample collection procedures to ensure data quality. Bridging studies are required to establish the correlation between plasma and DBS data to ensure that pooling of data from the various clinical studies can be used in population PK or PK/PD assessment. Seeking regulatory feedback and guidance on a case-by-case basis is recommended.

The purpose of this study is to discuss the surgical treatment for ovarian torsion in children and adolescents with a focus on the procedures of adnexal conservation surgery and its frequency in the literature of the last 10 years. We retrospectively reviewed the medical charts of 127 operative ovarian lesions including 30 ovarian torsions (23.6%) treated in two pediatric centers over a 10-year period. Age at presentation, presenting symptoms, diagnostic studies, surgical procedure and pathological findings were analyzed. Mean age was 13.7 years. Conservative surgery has been performed in 46.7% of the cases and laparoscopic approach in 40%. Ovarian torsion occurred in 56.7% on ovaries with functional lesion, in 23.3% on normal adnexa and in 20% on ovaries with benign neoplasm. The article includes a literature review (2000-2010) and a statistical analysis which shows a slow increase in conservative surgery from 28 to 45%. Laparoscopic surgery accounts for 23.5%. Literature review shows 40.5% normal adnexa, 33.2% non-neoplastic lesions, 25.3% benign neoplasms and 1% malignant neoplasms. The surgical treatment of children and adolescents presenting adnexal torsion should be practiced as an emergency and it should be more conservative as possible in order to maximize the future reproductive potential.

Anterior congenital urethrocutaneous fistula is a rare anomaly that may present in an isolated fashion or in association with other anomalies of the genital urinary tract or anorectal malformations. A case of congenital anterior urethrocutaneous fistula nonassociated with other congenital anomalies in a 3-year-old male whose mother has been exposed to Chernobyl's nuclear fallout is described. The patient was successfully operated with no recurrence. We report a review of the literature about etiology and surgical strategy including the role of ionizing radiations. The congenital anterior urethrocutaneous fistula represents a rare malformation. The etiopathogenesis is unknown.

Previous investigations in our laboratories resulted in the discovery of a novel series of potent nucleoside inhibitors of Hepatitis C virus (HCV) NS5B polymerase bearing tetracyclic 7-substituted 7-deaza-adenine nucleobases. The planarity of such modified systems was suggested to play a role in the high inhibitory potency observed. This paper describes how we envisaged to maintain the desired planarity of the modified nucleobase by means of an intra-molecular H-bond, engaging a H-bond donor atom on an appropriately substituted 7-heterocyclic residue with the adjacent amino group of the nucleobase. The success of this strategy is reflected by the identification of several novel potent nucleoside inhibitors of HCV NS5B bearing a 7-heterocyclic substituted 7-deaza-adenine nucleobase. Amongst these, the 1,2,4-oxadiazole analog 11 showed high antiviral potency against HCV replication in replicon cells and efficient conversion to the corresponding NTP in vivo, with high and sustained levels of NTP measured in rat liver following intravenous and oral administration., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

HCV NS3/4a protease inhibitors are proven therapeutic agents against chronic hepatitis C virus infection, with boceprevir and telaprevir having recently received regulatory approval as add-on therapy to pegylated interferon/ribavirin for patients harboring genotype 1 infections. Overcoming antiviral resistance, broad genotype coverage, and a convenient dosing regimen are important attributes for future agents to be used in combinations without interferon. In this communication, we report the preclinical profile of MK-5172, a novel P2-P4 quinoxaline macrocyclic NS3/4a protease inhibitor currently in clinical development. The compound demonstrates subnanomolar activity against a broad enzyme panel encompassing major hepatitis C virus (HCV) genotypes as well as variants resistant to earlier protease inhibitors. In replicon selections, MK-5172 exerted high selective pressure, which yielded few resistant colonies. In both rat and dog, MK-5172 demonstrates good plasma and liver exposures, with 24-h liver levels suggestive of once-daily dosing. When administered to HCV-infected chimpanzees harboring chronic gt1a or gt1b infections, MK-5172 suppressed viral load between 4 to 5 logs at a dose of 1 mg/kg of body weight twice daily (b.i.d.) for 7 days. Based on its preclinical profile, MK-5172 is anticipated to be broadly active against multiple HCV genotypes and clinically important resistance variants and highly suited for incorporation into newer all-oral regimens.

Purpose: The purpose of this study was to detect the role of serum tumor markers in the differential diagnosis and in the choice of the surgical treatment of ovarian lesions in pediatric age., Methods: We retrospectively reviewed medical charts of all pediatric girls operated in two pediatric centers during a 10-year period. The following features were analyzed: age at the moment of presentation, symptoms, diagnostic studies, surgical approach, pathological findings, and, in particular, serum tumor markers. Also, a literature review and statistical analysis (χ(2) test) concerning the increase of different tumor markers in benign and malignant ovarian lesions were related to the study., Results: A total of 120 children with 127 ovarian masses (7 with bilateral pathologies) were reviewed. Pathology showed 61 nonneoplastic lesions (48%), 54 benign tumors (42.5%), 5 malignant tumors (4%), and 7 torsed normal ovaries (5.5%). The evaluation of serum tumor markers was performed in 106 cases and was positive in 16 (15.1%): in all of the 5 cases (100%) of malignant neoplastic lesions and in 11 (20.4%) benign neoplastic ones. The literature review confirmed these data, and statistical analysis highlighted them as significant values., Conclusions: The role of tumor markers is still controversial. This is the reason why, before considering a radical treatment, we suggest caution to optimize future fertility.

A new class of HCV NS3/4a protease inhibitors containing a P2 to P4 macrocyclic constraint was designed using a molecular modeling-derived strategy. Building on the profile of previous clinical compounds and exploring the P2 and linker regions of the series allowed for optimization of broad genotype and mutant enzyme potency, cellular activity, and rat liver exposure following oral dosing. These studies led to the identification of clinical candidate 15 (MK-5172), which is active against genotype 1-3 NS3/4a and clinically relevant mutant enzymes and has good plasma exposure and excellent liver exposure in multiple species.

The clinical case described in this paper deals with a young female patient affected by primary hyperparathyroidism caused by an ectopic parathyroid adenoma of a supernumerary intrathymic parathyroid. The patient had hypercalcemia, in association with increased levels of parathormone, but was otherwise asymptomatic. Genetics tests for mutation of the MEN1, HRPT2, and CaSR genes were negative. She therefore underwent laboratory and instrumental tests but localization results in the neck were negative--only an intrathymic nodule was visualized. The complete surgical ablation of the thymus was conducted, which highlighted a nodule that, at histological examination, was shown to be an adenoma of a fifth parathyroid gland. The existence of a fifth, hyperfunctioning, intrathoracic parathyroid appears to be a rare cause of primary juvenile sporadic hyperparathyroidism. This peculiar clinical case could be of interest in similar cases evaluated by other surgeons.

As part of an ongoing medicinal chemistry effort to identify novel nucleoside inhibitors of HCV NS5B polymerase, we report the discovery of a novel series of 2'-C-Methyl-ribose nucleoside derivatives bearing a 7-aryl and 7-heteroaryl- substituted 7-deaza-adenine nucleobase. A reliable platform for the synthesis and simplified purification of the corresponding nucleoside triphosphates (NTPs) was established, enabling a solid understanding of the SAR relationship within the series. By this approach, we identified the novel analogs 13a and 13b that demonstrated micromolar levels of cellular activity, and the NTPs of which, 16a and 16b, are excellent inhibitors of NS5B with IC(50) = 0.1 μM, a level of intrinsic potency similar to that of previous and current clinical candidates.

The Hedgehog (Hh-) signaling pathway is a key developmental pathway which gets reactivated in many human tumors, and smoothened (Smo) antagonists are emerging as novel agents for the treatment of malignancies dependent on the Hh-pathway, with the most advanced compounds demonstrating encouraging results in initial clinical trials. A novel series of potent bicyclic hydantoin Smo antagonists was reported in the preceding article, these have been resolved, and optimized to identify potent homochiral derivatives with clean off-target profiles and good pharmacokinetic properties in preclinical species. While showing in vivo efficacy in mouse allograft models, unsubstituted bicyclic tetrahydroimidazo[1,5-a]pyrazine-1,3(2H,5H)-diones were shown to epimerize in plasma. Alkylation of the C-8 position blocks this epimerization, resulting in the identification of MK-5710 (47) which was selected for further development., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Compound A ((1aR,5S,8S,10R,22aR)-5-tert-butyl-N-{(1R,2S)-1-[(cyclopropylsulfonyl)carbamoyl]-2-ethenylcyclopropyl}-14-methoxy-3,6-dioxo-1,1a,3,4,5,6,9,10,18,19,20,21,22,22a-tetradecahydro-8H-7,10-methanocyclopropa[18,19][1,10,3,6]dioxadiazacyclononadecino[12,11-b]quinoline-8-carboxamide) is a prototype of a series of subnanomolar inhibitors of genotypes 1, 2, and 3 hepatitis C virus (HCV) NS3/4A proteases. HCV NS3/4A protease inhibitors have demonstrated high antiviral effects in patients with chronic HCV infection and are likely to form a key component of future HCV therapy. Compound A showed excellent liver exposure in rats, which is essential for compounds intended to treat HCV. The compound was mainly eliminated intact in bile and showed greater than dose proportional systemic exposure in rats. Compound A demonstrated time- and temperature-dependent uptake into rat and human hepatocytes and proved to be a substrate for rat hepatic uptake transporter Oatp1b2 and for human hepatic uptake transporters OATP1B1 and OATP1B3. The liver selectivity observed for this compound is likely to be due to transporter-mediated hepatic uptake together with moderate passive permeability. Metabolism was mainly CYP3A-mediated and generated a reactive epoxide on the vinylcyclopropyl sulfonamide moiety that could be quenched by glutathione. Similar metabolic profiles of Compound A were obtained in liver microsomes of rats and humans. The oral bioavailability at 5 mg/kg was low due to extensive hepatic first-pass effect but clearly the intestinal absorption was enough to deliver a high amount of the compound to the liver. The metabolism and disposition properties of Compound A are particularly attractive to support its evaluation as a drug candidate for the treatment of hepatitis C.

The purpose of this study was to investigate the in vivo absorption enhancement of a nucleoside (phosphoramidate prodrug of 2'-methyl-cytidine) anti-viral agent of proven efficacy by means of intestinal permeation enhancers. Natural nucleosides are hydrophilic molecules that do not rapidly penetrate cell membranes by diffusion and their absorption relies on specialized transporters. Therefore, the oral absorption of nucleoside prodrugs and the target organ concentration of the biologically active nucleotide can be limited due to poor permeation across the intestinal epithelium. In the present study, the specificity, concentration dependence, and effect of four classes of absorption promoters, i.e. fatty acids, steroidal detergents, mucoadhesive polymers, and secretory transport inhibitors, were evaluated in a rat in vivo model. Sodium caprate and alpha-tocopheryl-polyethyleneglycol-1000-succinate (TPGS) showed a significant effect in increasing liver concentration of nucleotide (5-fold). These results suggested that both excipients might be suited in a controlled release matrix for the synchronous release of the drug and absorption promoter directly to the site of absorption and highlights that the effect is strictly dependent on the absorption promoter dose. The feasibility of such a formulation approach in humans was evaluated with the aim of developing a solid dosage form for the peroral delivery of nucleosides and showed that these excipients do provide a potential valuable tool in pre-clinical efficacy studies to drive discovery programs forward.

A novel strategy to minimize phospholipids-based matrix effects in bioanalytical LC-MS/MS assays was evaluated. The phospholipids-based matrix effect was investigated with a commercially available electrospray ionization (ESI) source coupled with a triple quadrupole mass spectrometer. A systematic comparison approach of two sample preparation procedures was performed. In particular, the matrix effect on mass spectrometry response in rat and human plasma samples was studied by comparing sample extracts obtained by means of a conventional plasma protein precipitation with acetonitrile and the novel HybridSPE-Precipitation procedure. The HybridSPE dramatically reduced the levels of residual phospholipids in biological samples, leading to significant reduction in matrix effects. This new procedure which combines the simplicity of precipitation with the selectivity of SPE allows to obtain much cleaner extracts than with conventional procedures. The effective targeted removal of phospholipids and proteins in biological plasma samples achieved with the HybridSPE-Precipitation procedure provides significant improvement in bioanalysis and a practical and fast way to ensure the avoidance of phospholipids-based matrix effects.

The application of a phosphoramidate prodrug approach to 2'-C-methylcytidine (NM107), the first nucleoside inhibitor of the hepatitis C virus (HCV) NS5B polymerase, is reported. 2'-C-Methylcytidine, as its valyl ester prodrug (NM283), was efficacious in reducing the viral load in patients infected with HCV. Several of the phosphoramidates prepared demonstrated a 10- to 200-fold superior potency with respect to the parent nucleoside in the cell-based replicon assay. This is due to higher levels of 2'-C-methylcytidine triphosphate in the cells. These prodrugs are efficiently activated and converted to the triphosphate in hepatocytes of several species. Our SAR studies ultimately led to compounds that gave high levels of NTP in hamster and rat liver after subcutaneous dosing and that were devoid of the toxic phenol moiety usually found in ProTides.

The currently approved treatment for hepatitis C virus infections is a combination of Ribavirin and pegylated Interferon. It leads to a sustained virologic response in approximately only half of the patients treated. For this reason there is an urgent need of new therapeutic agents. 2'-C-Methylcytidine is the first nucleoside inhibitor of the HCV NS5B polymerase that was efficacious in reducing the viral load in patients infected with HCV. The application of a monophosphate prodrug approach based on unprecedented cyclic phosphoramidates is reported. Our SAR studies led to compounds that are efficiently converted to the active triphosphate in human hepatocytes.

A very accurate and selective LC-MS/MS method was developed and validated for the quantification of 2'-C-modified nucleoside triphosphate in liver tissue samples. An efficient pretreatment procedure of liver tissue samples was developed, using a fully automated SPE procedure with 96-well SPE plate (weak anion exchange sorbent, 30 mg). Nucleotide hydrophilic interaction chromatography has been performed on an aminopropyl column (100 mm x 2.0 mm, 3 microm) using a gradient mixture of ACN and ACN/water (5:95 v/v) with 20 mM ammonium acetate at pH 9.45 as mobile phase at 300 microL/min flow rate. The 2'-C-modified nucleoside triphosphate was detected in the negative ESI mode in multiple reaction monitoring (MRM) mode. Calibration curve was linear over the 0.05-50 microM concentration range. Satisfying results, confirming the high reliability of the established LC-MS/MS method, were obtained for intraday precision (CV = 2.5-9.1%) and accuracy (92.6-94.8%) and interday precision (CV = 9.6-11.5%) and accuracy (94.4-102.4%) as well as for recovery (82.0-112.6%) and selectivity. The method has been successfully applied for pharmacokinetic studies of 2'-C-methyl-cytidine-triphosphate in liver tissue samples.

A variety of new prodrugs of 2'-methyl cytidine based on acyloxy ethylamino phosphoramidates have been synthesized and tested in vitro and in vivo for their biological activity. Compared with the parent drug a 10- to 20-fold increase in formation of nucleotide triphosphate in rat and human hepatocytes could be achieved.

Purpose: To study optical aberrations in eyes having uneventful cataract surgery and in-the-bag implantation of an aspherical Tecnis Z9000 intraocular lens (IOL) (AMO) or a spherical CeeOn Edge 911 IOL (Pharmacia) and compare the results with those in a group of phakic age-matched eyes., Setting: Ophthalmic Unit, Hospital and University of Verona, Verona, Italy., Materials: Three groups, each with 30 patients (30 eyes), were examined with the Topcon KR-9000PW topographer/aberrometer. Spherical aberration and coma were analyzed in detail for 4.0 mm optical zone. Internal values were obtained by subtracting corneal aberrations from ocular aberrations. Point spread function and modulation transfer function (MTF) were considered for optical quality., Results: The mean internal spherical aberration Z(4)(0) was -0.048 microm +/- 0.017 (SD) in the Tecnis group, +0.033 +/- 0.026 microm in the 911 Edge group (P<.001), and -0.013 +/- 0.056 microm in the phakic group (P = .149). The mean internal vertical coma Z(3)(-1) was 0.087 +/- 0.056 microm, 0.054 +/- 0.043 microm (P = .005), and 0.044 +/- 0.044 microm (P<.001), respectively. The internal horizontal coma Z(3)(+1) showed a similar pattern; however, total resulting coma was similar in the 3 groups. The mean Strehl ratio was 0.284 +/- 0.166 in the Tecnis group, 0.145 +/- 0.077 in the 911 Edge group (P<.01), and 0.164 +/- 0.097 in the phakic group (P<.01). The MTF curve was better in the Tecnis group (P<.001)., Conclusions: After uneventful implantation, the aspherical IOL yielded better ocular aberration and optical quality results than the spherical IOL. Induced coma was somewhat higher in the Tecnis group; however, the overall results were not affected. Physiologic IOL decentration after correct in-the-bag implantation did not negate the advantages of asphericity.

Accurate mass measurements are used to determine the elemental composition and formulae of molecules to confirm their identity or to assist in their characterization. Currently, the most widely used techniques for measuring exact masses employ magnetic sector instruments, Fourier transform ion cyclotron resonance mass spectrometers and lower resolution instruments such as time-of-flight (TOF) and quadrupole-TOF. This paper reports the accurate mass measurement using a triple quadrupole mass spectrometer. Indeed, the recently introduced triple quadrupole mass spectrometer, with unique enhanced mass-resolution capability, has demonstrated simple data acquisition methods and requires few experiments to measure exact masses with accuracy and determines elemental compositions of both protonated and deprotonated molecules. All the accurate mass measurements were performed using both positive and negative electrospray ionization in enhanced mass-resolution mode (peak width of 0.1 Th FWMH). Several new drug entities were investigated as simulated unknowns and analyzed by means of an accurate mass liquid chromatography/electrospray ionization mass spectrometry (AM-LC/ESI-MS) method. The accurate mass measurements resulted in only one proposed elemental composition for all tested compounds, using reasonable elemental limits and mass tolerance for the calculation. Moreover, all the experimentally determined accurate mass measurements gave satisfactory results in terms of accuracy (lower than 5 ppm)., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

A very highly sensitive and highly selective liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method was developed to evaluate and quantify a new drug candidate in different biological matrices. Following a simple plasma protein precipitation using acetonitrile, the post-treatment samples were analyzed on a C18 column interfaced with a new generation of triple-quadrupole mass spectrometer. The recently introduced triple-quadrupole mass spectrometer, the TSQ Quantum Ultra, with enhanced mass-resolution capability, demonstrated improved sensitivity (0.05 ng/mL), coupled with suitable accuracy and precision, over a broad linear dynamic range (0.05-1000 ng/mL). A comparison of the assay performance data (dynamic range, calibration curve equation, precision and accuracy) of the enhanced resolution method against a unit resolution method under optimized conditions showed the performance improvement of the enhanced mass resolution method for bioanalytical high-throughput applications. The enhanced mass resolution method herein described was successfully applied to the evaluation of the pharmacokinetic profile of a new drug candidate in rat, rabbit and dog plasma samples., (Copyright 2006 John Wiley & Sons, Ltd.)