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Background: To address multidrug resistance, we developed engineered Cationic Antimicrobial Peptides (eCAPs). Lead eCAP WLBU2 displays potent activity against drug-resistant bacteria and effectively treats lethal bacterial infections in mice, reducing bacterial loads to undetectable levels in diverse organs. Objective: To support development of WLBU2, we conducted a mass balance study. Methods: CD1 mice were administered 10, 15, 20 and 30 mg/kg of QDx5 WLBU2 or a single dose of [14C]-WLBU2 at 15 mg/kg IV. Tolerability, tissue distribution and excretion were evaluated with liquid scintillation and HPLC-radiochromatography. Results: The maximum tolerated dose of WLBU2 is 20 mg/kg IV. We could account for greater than >96% of the radioactivity distributed within mouse tissues at 5 and 15 min. By 24h, only ~40-50% of radioactivity remained in the mice. The greatest % of the dose was present in liver, accounting for ~35% of radioactivity at 5 and 15 min, and ~ 8% of radioactivity remained at 24h. High radioactivity was also present in kidneys, plasma, red blood cells and lungs, while less than 0.2% of radioactivity was present in brain, fat, or skeletal muscle. Urinary and fecal excretion accounted for 12.5 and 2.2% of radioactivity at 24h. Conclusion: WLBU2 distributes widely to mouse tissues and is rapidly cleared with a terminal radioactivity half-life of 22 h, a clearance of 27.4 mL/h/kg, and a distribution volume of 0.94 L/kg. At 2-100 μg-eq/g, the concentrations of 14C-WLBU2 appear high enough in the tissues to account for the inhibition of microbial growth.

To evaluate the effect of demographic disparities on language outcomes in a diverse group of children who are deaf or hard of hearing.Retrospective cohort study.UCSF Benioff Children's Hospital (a tertiary care center).Forty-four patients aged18 years were identified with sensorineural hearing loss managed with a behind-the-ear hearing aid or cochlear implant. Demographic and clinical data were extracted from the medical record. The primary outcome measure was the Preschool Language Scales-5 at least 6 months after intervention. Predictors of language outcome were assessed: hearing level at the time of hearing intervention, cochlear implant status, age of identification and intervention, travel time to site of hearing care, home language, race/ethnicity, insurance type, and Access Challenge Index-a novel measure of educational environment and family support based on the Child Cochlear Implant Profile. Multivariate and univariate analysis assessed predictors for association with intervention and receptive, expressive, and total language scores.Overall 82% of patients had cochlear implants. The median age at hearing intervention was 12 months. The sample was 59% female, 52% non-White, and 61% publicly insured, and 20% had a non-English primary home language. Accounting for multiple demographic and clinical predictors, a high Access Challenge Index score was independently associated with longer time to intervention (Access Challenge Index-a novel comprehensive measure of educational and family environment-is a strong independent predictor of language outcomes in children who are deaf or hard of hearing.

Purpose The purpose of this study was to determine the incidence of minor and major ocular injuries in patients with orbital wall fractures at Loma Linda University Health, a level-one trauma center, to help determine the most appropriate setting for the initial dilated fundus examination by ophthalmologists. Methods A retrospective study was performed from January 2008 to January 2013 of patients diagnosed with orbital wall fracture secondary to trauma. Exclusion criteria included unknown mechanism of injury, the absence of ophthalmology consultation, or absence of imaging. Data collected included age, gender, mechanism of injury, visual acuity, and anterior/posterior segment findings. Ocular injuries were categorized as either minor or major. Results Of 567 charts reviewed, 460 met criteria and were included for analysis. In the analysis, 86.5% of patients were male, and 81.3% were Caucasian. The most common mechanism of orbital fracture was blunt injury. Visual acuity was better than 20/100 in 82.4% of patients. On chart review, 81.1% of patients were found to have either a minor injury, a major injury, or both. The most common injury was subconjunctival hemorrhage (53.5%). Globe rupture (2.9%) and vision-threatening posterior segment findings such as retinal tear and choroidal rupture (1.3%) were relatively rare. Only one retinal detachment (0.2%) was found, specifically in the setting of severe injury with concomitant globe rupture. Conclusion Knowledge of the common ocular injuries associated with orbital fractures will help emergency department (ED) physicians and ophthalmologists provide the dilated fundus exam in the most appropriate setting. The most frequent injuries identified were non-vision threatening, and visually significant posterior segment findings were relatively rare (1.3%). Thus, for the majority of patients presenting to the ED with orbital fracture, a dilated fundus exam can be performed at a later date in the outpatient clinic setting, unless urgent orbital fracture surgery is planned.

PURPOSE To update the incidence of orbital fractures in U.S. Soldiers admitted to the former Walter Reed Army Medical Center from 2001 to 2011 after sustaining combat injuries in Operation Iraqi Freedom and Operation Enduring Freedom. METHODS Data were collected in the Walter Reed Ocular Trauma Database. Inclusion criteria were any U.S. Soldier or Department of Defense civilian with an orbital fracture injured in Operation Iraqi Freedom/Operation Enduring Freedom. Primary outcome measures were final visual acuity and the effect of orbital fracture, number of fractures, and anatomic location of fracture on final visual acuity. RESULTS Eight-hundred ninety eye injuries occurred in 652 patients evacuated to Walter Reed Army Medical Center between 2001 and 2011. Orbital fractures occurred in 304 eyes (34.2%). A single wall was fractured in 140 eyes (46.05%), 2 in 99 (32.6%) eyes, 3 in 31 (10.2%), 4 in 28 (9.2%), and unknown in 6 (1.9%) eyes. Roof fractures were found in 74 (24.34%), medial wall in 135 (44.41%), lateral wall in 109 (35.9%), and floor fractures in 217 (71.4%). Final visual acuity was analyzed and 140 (46.05%) eyes had greater than 20/40 vision, 17 (5.59%) were 20/50 to 20/200, 26 (8.5%) were count fingers to light perception, and 95 (31.3%) were no light perception. In logistic regression analysis, roof (p = 0.001), medial (p = 0.009), and lateral fractures (p = 0.016) were significantly associated with final visual acuity less than 20/200, while floor fractures were not (p = 0.874). Orbital fracture and all fracture subtypes were significantly associated with traumatic brain injury, retrobulbar hematoma, optic nerve injury, but not for vitreous hemorrhage, commotio, hyphema, and choroidal rupture. Fracture repair was noted in 45 (14.8%). CONCLUSIONS Orbital fractures occurred in a third of Operation Iraqi Freedom/Operation Enduring Freedom eyes of ocular trauma patients referred to one tertiary care military hospital. This resulted in approximately 40% of these eyes remaining legally blind after injury.Orbital fractures occur commonly during combat trauma and a significant number are legally blind despite appropriate treatment.

Systematic unbiased high-throughput screening (HTS) of drug combinations (DCs) in well-characterized tumor cell lines is a data-driven strategy to identify novel DCs with potential to be developed into effective therapies. Four DCs from a DC HTS campaign were selected for confirmation; only one appears in clinicaltrials.gov and limited preclinical in vitro data indicates that the drug pairs interact synergistically. Nineteen DC-tumor cell line sets were confirmed to interact synergistically in three pharmacological interaction models. We developed an imaging assay to quantify accumulation of the ABCG2 efflux transporter substrate Hoechst. Gefitinib and raloxifene enhanced Hoechst accumulation in ABCG2 (BCRP)-expressing cells, consistent with inhibition of ABCG2 efflux. Both drugs also inhibit ABCB1 efflux. Mitoxantrone, daunorubicin, and vinorelbine are substrates of one or more of the ABCG2, ABCB1, or ABCC1 efflux transporters expressed to varying extents in the selected cell lines. Interactions between ABC drug efflux transporter inhibitors and substrates may have contributed to the observed synergy; however, other mechanisms may be involved. Novel synergistic DCs identified by HTS were confirmed in vitro, and plausible mechanisms of action studied. Similar approaches may justify the testing of novel HTS-derived DCs in mouse xenograft human cancer models and support the clinical evaluation of effective in vivo DCs in patients.

Background. COVID-19 may result in multiorgan failure and death. Early detection of patients at risk may allow triage and more intense monitoring. The aim of this study was to develop a simple, objective admission score, based on laboratory tests, that identifies patients who are likely going to deteriorate. Methods. This is a retrospective cohort study of all COVID-19 patients admitted to a tertiary academic medical center in New York City during the COVID-19 crisis in spring 2020. The primary combined endpoint included intubation, stage 3 acute kidney injury (AKI), or death. Laboratory tests available on admission in at least 70% of patients (and age) were included for univariate analysis. Tests that were statistically or clinically significant were then included in a multivariate binary logistic regression model using stepwise exclusion. 70% of all patients were used to train the model, and 30% were used as an internal validation cohort. The aim of this study was to develop and validate a model for COVID-19 severity based on biomarkers. Results. Out of 2545 patients, 833 (32.7%) experienced the primary endpoint. 53 laboratory tests were analyzed, and of these, 47 tests (and age) were significantly different between patients with and without the endpoint. The final multivariate model included age, albumin, creatinine, C-reactive protein, and lactate dehydrogenase. The area under the ROC curve was 0.850 (CI [95%]: 0.813, 0.889), with a sensitivity of 0.800 and specificity of 0.761. The probability of experiencing the primary endpoint can be calculated as p = e − 2.4475 + 0.02492 age − 0.6503 albumin + 0.81926 creat + 0.00388 CRP + 0.00143 LDH / 1 + e − 2.4475 + 0.02492 age − 0.6503 albumin + 0.81926 creat + 0.00388 CRP + 0.00143 LDH . Conclusions. Our study demonstrated that poor outcome in COVID-19 patients can be predicted with good sensitivity and specificity using a few laboratory tests. This is useful for identifying patients at risk during admission.

BACKGROUND: Critically ill coronavirus disease 2019 (COVID-19) patients have frequent thrombotic complications and laboratory evidence of hypercoagulability. The relationship of coagulation tests and thrombosis requires investigation to identify best diagnostic and treatment approaches. We assessed for hypercoagulable characteristics in critically ill COVID-19 patients using rotational thromboelastometry (ROTEM) and explored relationships of D-dimer and ROTEM measurements with thrombotic complications. METHODS: Critically ill adult COVID-19 patients receiving ROTEM testing between March and April 2020 were analyzed. Patients receiving therapeutic anticoagulation before ROTEM were excluded. Rotational thromboelastometry measurements from COVID-19 patients were compared with non−COVID-19 patients matched by age, sex, and body mass index. Intergroup differences in ROTEM measurements were assessed using t tests. Correlations of D-dimer levels to ROTEM measurements were assessed in COVID-19 patients who had available concurrent testing. Intergroup differences of D-dimer and ROTEM measurements were explored in COVID-19 patients with and without thrombosis. RESULTS: Of 30 COVID-19 patients receiving ROTEM, we identified hypercoagulability from elevated fibrinogen compared with non- COVID-19 patients (fibrinogen assay maximum clot firmness [MCF], 47 ± 13 mm vs. 20 ± 7 mm; mean intergroup difference, 27.4 mm; 95% confidence interval [CI], 22.1−32.7 mm; p < 0.0001). In our COVID-19 cohort, thrombotic complications were identified in 33%. In COVID-19 patients developing thrombotic complications, we identified higher D-dimer levels (17.5 ± 4.3 qg/mL vs. 8.0 ± 6.3 qg/mL; mean difference, 9.5 qg/mL; 95% CI, 13.9−5.1; p < 0.0001) but lower fibrinogen assay MCF (39.7 ± 10.8 mm vs. 50.1 ± 12.0 mm; mean difference, −11.2 mm; 95% CI, −2.1 to −20.2; p = 0.02) compared with patients without thrombosis. We identified negative correlations of D-dimer levels and ROTEM MCF in these patients (r = −0.61; p = 0.001). CONCLUSION: We identified elevated D-dimer levels and hypercoagulable blood clot characteristics from increased fibrinogen on ROTEM testing in critically ill COVID-19 patients. However, we identified lower, albeit still hypercoagulable, ROTEM measurements offibrino- gen in COVID-19 patients with thrombotic complications compared with those without. Further work is required to externally validate these findings and to investigate the mechanistic drivers for these relationships to identify best diagnostic and treatment approaches for these patients. LEVEL OF EVIDENCE: Epidemiologic, level IV.

COVID-19 has created an enormous health crisis and this spring New York City had a severe outbreak that pushed health and critical care resources to the limit. A lack of adequate space for mechanically ventilated patients induced our hospital to convert operating rooms into critical care areas (OR-ICU). A large number of COVID-19 will develop acute kidney injury that requires renal replacement therapy (RRT). We included 116 patients with COVID-19 who required mechanical ventilation and were cared for in our OR-ICU. At 90 days and at discharge 35 patients died (30.2%). RRT was required by 45 of the 116 patients (38.8%) and 18 of these 45 patients (40%) compared to 17 with no RRT (23.9%, ns) died during hospitalization and after 90 days. Only two of the 27 patients who required RRT and survived required RRT at discharge and 90 days. When defining renal recovery as a discharge serum creatinine within 150% of baseline, 68 of 78 survivors showed renal recovery (87.2%). Survival was similar to previous reports of patients with severe COVID-19 for patients cared for in provisional ICUs compared to standard ICUs. Most patients with severe COVID-19 and AKI are likely to recover full renal function., Highlights • We retrospectively assessed renal outcomes and mortality in 116 patients with severe COVID-19 who were cared for in a provisional OR ICU. • A large number of these patients will develop acute kidney injury: Stage 1 AKI: 20.9%, Stage 2 AKI: 14.8, Stage 3 AKI: 31.9%. • The overall mortality was 30.2%. • 38.8% required renal replacement therapy but only 2 of the survivors required renal replacement therapy at discharge and at 90 day after admission and 68 of 78 survivors showed renal recovery (87.2%). • Acute kidney injury is common with severe COVID-19 but most survivors are likely to recover full renal function.

Hedgehog signaling is essential for bone formation, including functioning as a means for the growth plate to drive skeletal mineralization. However, the mechanisms regulating hedgehog signaling specifically in bone-forming osteoblasts are largely unknown. Here, we identified SLIT and NTRK-like protein-5(Slitrk5), a transmembrane protein with few identified functions, as a negative regulator of hedgehog signaling in osteoblasts. Slitrk5 is selectively expressed in osteoblasts and loss of Slitrk5 enhanced osteoblast differentiation in vitro and in vivo. Loss of SLITRK5 in vitro leads to increased hedgehog signaling and overexpression of SLITRK5 in osteoblasts inhibits the induction of targets downstream of hedgehog signaling. Mechanistically, SLITRK5 binds to hedgehog ligands via its extracellular domain and interacts with PTCH1 via its intracellular domain. SLITRK5 is present in the primary cilium, and loss of SLITRK5 enhances SMO ciliary enrichment upon SHH stimulation. Thus, SLITRK5 is a negative regulator of hedgehog signaling in osteoblasts that may be attractive as a therapeutic target to enhance bone formation., Hedgehog signaling is essential for bone formation. Here, the authors show that the transmembrane protein SLITRK5 is a negative regulator of hedgehog signaling in osteoblasts, suggesting it may be a potential therapeutic target to enhance bone formation.

Objective To characterize patients with coronavirus disease 2019 (covid-19) in a large New York City medical center and describe their clinical course across the emergency department, hospital wards, and intensive care units. Design Retrospective manual medical record review. Setting NewYork-Presbyterian/Columbia University Irving Medical Center, a quaternary care academic medical center in New York City. Participants The first 1000 consecutive patients with a positive result on the reverse transcriptase polymerase chain reaction assay for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) who presented to the emergency department or were admitted to hospital between 1 March and 5 April 2020. Patient data were manually abstracted from electronic medical records. Main outcome measures Characterization of patients, including demographics, presenting symptoms, comorbidities on presentation, hospital course, time to intubation, complications, mortality, and disposition. Results Of the first 1000 patients, 150 presented to the emergency department, 614 were admitted to hospital (not intensive care units), and 236 were admitted or transferred to intensive care units. The most common presenting symptoms were cough (732/1000), fever (728/1000), and dyspnea (631/1000). Patients in hospital, particularly those treated in intensive care units, often had baseline comorbidities including hypertension, diabetes, and obesity. Patients admitted to intensive care units were older, predominantly male (158/236, 66.9%), and had long lengths of stay (median 23 days, interquartile range 12-32 days); 78.0% (184/236) developed acute kidney injury and 35.2% (83/236) needed dialysis. Only 4.4% (6/136) of patients who required mechanical ventilation were first intubated more than 14 days after symptom onset. Time to intubation from symptom onset had a bimodal distribution, with modes at three to four days, and at nine days. As of 30 April, 90 patients remained in hospital and 211 had died in hospital. Conclusions Patients admitted to hospital with covid-19 at this medical center faced major morbidity and mortality, with high rates of acute kidney injury and inpatient dialysis, prolonged intubations, and a bimodal distribution of time to intubation from symptom onset.

ObjectiveTo characterize patients with coronavirus disease 2019 (COVID-19) in a large New York City (NYC) medical center and describe their clinical course across the emergency department (ED), inpatient wards, and intensive care units (ICUs).DesignRetrospective manual medical record review.SettingNewYork-Presbyterian/Columbia University Irving Medical Center (NYP/CUIMC), a quaternary care academic medical center in NYC.ParticipantsThe first 1000 consecutive patients with laboratory-confirmed COVID-19.MethodsWe identified the first 1000 consecutive patients with a positive RT-SARS-CoV-2 PCR test who first presented to the ED or were hospitalized at NYP/CUIMC between March 1 and April 5, 2020. Patient data was manually abstracted from the electronic medical record.Main outcome measuresWe describe patient characteristics including demographics, presenting symptoms, comorbidities on presentation, hospital course, time to intubation, complications, mortality, and disposition.ResultsAmong the first 1000 patients, 150 were ED patients, 614 were admitted without requiring ICU-level care, and 236 were admitted or transferred to the ICU. The most common presenting symptoms were cough (73.2%), fever (72.8%), and dyspnea (63.1%). Hospitalized patients, and ICU patients in particular, most commonly had baseline comorbidities including of hypertension, diabetes, and obesity. ICU patients were older, predominantly male (66.9%), and long lengths of stay (median 23 days; IQR 12 to 32 days); 78.0% developed AKI and 35.2% required dialysis. Notably, for patients who required mechanical ventilation, only 4.4% were first intubated more than 14 days after symptom onset. Time to intubation from symptom onset had a bimodal distribution, with modes at 3-4 and 9 days. As of April 30, 90 patients remained hospitalized and 211 had died in the hospital.ConclusionsHospitalized patients with COVID-19 illness at this medical center faced significant morbidity and mortality, with high rates of AKI, dialysis, and a bimodal distribution in time to intubation from symptom onset.

Made available in DSpace on 2018-11-26T15:45:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-11-30 NCI-CTEP NCI Institute of International Education Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The ribonucleotide reductase inhibitor and radiosensitizer triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP), NSC 663249) is clinically being evaluated via the intravenous (IV) route for the treatment of cervical and vulvar cancer in combination with primary cisplatin chemoradiation. The need for a 2-h infusion and frequent administration of triapine is logistically challenging, prompting us to pursue oral (PO) administration. In support of the clinical trial investigating oral triapine in combination with chemoradiation, we developed and validated a novel LC-MS/MS assay for the quantification of triapine in 50 mu L human plasma. After protein precipitation, chromatographic separation of the supernatant was achieved with a Shodex ODP2 column and an isocratic acetonitrile-water mobile phase with 10% ammonium acetate. Detection with an ABI 4000 mass spectrometer utilized electrospray positive mode ionization. The assay was linear from 3 to 3,000 ng/mL and proved to be accurate (97.1-103.1%) and precise (

The c-Jun N-terminal kinases (JNKs) are ancient and evolutionarily conserved regulators of proliferation, differentiation, and cell death responses. Currently, in vitro studies offer conflicting data about whether the JNK pathway augments or represses osteoblast differentiation, and the contribution of the JNK pathway to regulation of bone mass in vivo remains unclear. Here we show that Jnk1-/- mice display severe osteopenia due to impaired bone formation, whereas Jnk2-/- mice display a mild osteopenia only evident in long bones. In order to both confirm that these effects were osteoblast intrinsic and assess whether redundancy with JNK1 masks a potential contribution of JNK2, mice with a conditional deletion of both JNK1 and JNK2 floxed conditional alleles in osteoblasts (Jnk1-2osx ) were bred. These mice displayed a similar degree of osteopenia to Jnk1-/- mice due to decreased bone formation. In vitro, Jnk1-/- osteoblasts display a selective defect in the late stages of osteoblast differentiation with impaired mineralization activity. Downstream of JNK1, phosphorylation of JUN is impaired in Jnk1-/- osteoblasts. Transcriptome analysis showed that JNK1 is required for upregulation of several osteoblast-derived proangiogenic factors such as IGF2 and VEGFa. Accordingly, JNK1 deletion results in a significant reduction skeletal vasculature in mice. Taken together, this study establishes that JNK1 is a key mediator of osteoblast function in vivo and in vitro. © 2017 American Society for Bone and Mineral Research.

Background Reliable detection of slowed vertical saccades may help discriminate progressive supranuclear palsy (PSP) from the subset of Parkinson’s disease patients who lack tremor (akinetic-rigid or PD-postural instability and gait disorder PIGD subtype), and from age-related oculomotor changes. We investigated the feasibility of a camera-less computerized behavioral saccade latency paradigm previously validated in PD to discriminate probable PSP-Richardson syndrome (PSP-RS) from PD-PIGD and age-matched controls. Methods In this proof-of-concept case-control study, reflexive saccade latencies were measured in 5 subjects with probable PSP-RS, 5 subjects with PD-PIGD subtype, and 5 age-matched controls using the behavioral paradigm. The battery was repeated approximately one month later. All subjects were examined off levodopa by a movement disorders neurologist and by an ophthalmologist, who also performed a dilated eye exam. Results Vertical prosaccade latencies were longer in the PSP group (median = 903 ms) relative to PD (median = 268 ms) and control groups (median = 235 ms), with no overlap between groups (100% accuracy). PSP subjects also had larger vertical-horizontal discrepancies than comparison groups. Test-retest reliability for the behavioral saccade measures was good (interclass correlation coefficient = 0.948; 95% confidence interval [0.856, 0.982]), and the measures strongly correlated with clinical ratings. Conclusions Computerized behavioral measurement of reflexive saccade latency is feasible in PSP, and potentially discriminates probable PSP-RS from the PD-PIGD subtype. Findings from this proof-of-concept study support utility of the approach for obtaining objective saccade metrics in clinical evaluations and for tracking change in future, larger trials of moderately advanced PSP. Future studies should also examine the behavioral paradigm in earlier presentations of PSP and other subtypes of PSP.

PurposeThe oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) promotes gene transcription involved in cancer, and its activation by IL-6 is found in head and neck squamous cell carcinoma. Four triazolothiadizine STAT3 pathway inhibitors were evaluated to prioritize a single compound for in vivo examination.MethodsMetabolic stability in mouse liver microsome incubation was used to evaluate four triazolothiadizine analogues, and UPCDC-10205 was administered to mice IV as single or multiple doses to evaluate toxicity. Single-dose pharmacokinetics (PK), bioavailability and metabolism were studied after IV 4mg/kg, PO 4mg/kg, or PO 30mg/kg suspension in 1% carboxymethyl cellulose. Mice were euthanized between 5min to 24h after dosing, and plasma and tissues were analyzed by LC-MS. Non-compartmental PK parameters were determined.ResultsOf the four triazolothiadizine analogues evaluated, UPCDC-10205 was metabolically most stable. The maximum soluble dose of 4mg/kg in 10% Solutol™ was not toxic to mice after single and multiple doses. PK analysis showed extensive tissue distribution and rapid plasma clearance. Bioavailability was ~5%. A direct glucuronide conjugate was identified as the major metabolite which was recapitulated in vitro.ConclusionsRapid clearance of UPCDC-10205 was thought to be the result of phase II metabolism despite its favorable stability in a phase I in vitro metabolic stability assay. The direct glucuronidation explains why microsomal stability (reflective of phase I metabolism) did not translate to in vivo metabolic stability. UPCDC-10205 did not demonstrate appropriate exposure to support efficacy studies in the current formulation.

Indenoisoquinolines are non-camptothecin topoisomerase I (TopI) inhibitors that overcome the limitations of camptothecins: chemical instability and camptothecin resistance. Two dosing schedules of the novel indenoisoquinoline, indotecan (LMP400), were evaluated in patients with advanced solid tumors. The maximum tolerated dose (MTD), toxicities, and pharmacokinetics of two indotecan drug administration schedules (daily for 5 days or weekly) were investigated. Modulation of TopI and the phosphorylation of histone H2AX (γH2AX) were assayed in tumor biopsies; γH2AX levels were also evaluated in circulating tumor cells (CTCs) and hair follicles to assess DNA damage response. An MTD of 60 mg/m2/day was established for the daily regimen, compared to 90 mg/m2 for the weekly regimen. The TopI response to drug showed target engagement in a subset of tumor biopsies. Pharmacokinetics profiles demonstrated a prolonged terminal half-life and tissue accumulation compared to topotecan. Dose-dependent decreases in total CTCs were measured in seven patients. Formation of γH2AX-positive foci in CTCs (day 3) and hair follicles (4–6 h) was observed following treatment. We established the MTD of two dosing schedules for a novel TopI inhibitor, indotecan. Target engagement was demonstrated as Top1 downregulation and γH2AX response. No objective responses were observed on either schedule in this small patient cohort. The principal toxicity of both schedules was myelosuppression; no significant gastrointestinal problems were observed. Increased DNA damage response was observed in CTCs, hair follicles, and a subset of tumor biopsies.

The present study is the first to report inhibition of breast cancer cell growth in vitro and in vivo and suppression of self-renewal of breast cancer stem cells (bCSC) by a pine bark component (leelamine). Except for a few recent publications in melanoma, anticancer pharmacology of this interesting phytochemical is largely elusive. Leelamine (LLM) dose-dependently inhibited viability of MDA-MB-231 (triple-negative), MCF-7 (estrogen receptor-positive), and SUM159 (triple-negative) human breast cancer cells in association with apoptotic cell death induction. To the contrary, a normal mammary epithelial cell line derived from fibrocystic breast disease and spontaneously immortalized (MCF-10A) was fully resistant to LLM-mediated cell growth inhibition and apoptosis induction. LLM also inhibited self-renewal of breast cancer stem cells. Apoptosis induction by LLM in breast cancer cells was accompanied by a modest increase in reactive oxygen species production, which was not due to inhibition of mitochondrial electron transport chain complexes. Nevertheless, ectopic expression of manganese superoxide dismutase conferred partial protection against LLM-induced cell death but only at a lower yet pharmacologically relevant concentration. Exposure of breast cancer cells to LLM resulted in (a) induction and/or activation of multidomain proapoptotic proteins Bax and Bak, (b) caspase-9 activation, and (c) cytosolic release of cytochrome c. Bax and Bak deficiency in immortalized fibroblasts conferred significant protection against cell death by LLM. Intraperitoneal administration of LLM (7.5 mg/kg; 5 times/wk) suppressed the growth of orthotopic SUM159 xenografts in mice without any toxicity. In conclusion, the present study provides critical preclinical data to warrant further investigation of LLM. © 2016 Wiley Periodicals, Inc.

Background: Cervical cancer is the second most common cancer in women worldwide [1]. Photodynamic therapy has been used for cervical intraepithelial neoplasia with good responses, but few studies have used newer phototherapeutics. We evaluated the effectiveness of photodynamic therapy using Pc 4 in vitro and in vivo against human cervical cancer cells. Methods: CaSki and ME-180 cancer cells were grown as monolayers and spheroids. Cell growth and cytotoxicity were measured using a methylthiazol tetrazolium assay. Pc 4 cellular uptake and intracellular distribution were determined. For in vitro Pc 4 photodynamic therapy, cells were irradiated at 667 nm at a fluence of 2.5 J/cm2 at 48 h. SCID mice were implanted with CaSki and ME-180 cells both subcutaneously and intracervically. Forty-eight hours after Pc 4 photodynamic therapy was administered at 75 and 150 J/cm2. Results: The IC50s for Pc 4 and Pc 4 photodynamic therapy for CaSki and ME-180 cells as monolayers were, 7.6 μM and 0.016 μM and >10 μM and 0.026 μM; as spheroids, IC50s of Pc 4 photodynamic therapy were, 0.26 μM and 0.01 μM. Pc 4 was taken up within cells and widely distributed in tumors and tissues. Intracervical photodynamic therapy resulted in tumor death, however mice died due to gastrointestinal toxicity. Photodynamic therapy resulted in subcutaneous tumor death and growth delay. Conclusions: Pc 4 photodynamic therapy caused death within cervical cancer cells and xenografts, supporting development of Pc 4 photodynamic therapy for treatment of cervical cancer. Support: P30-CA47904, CTSI BaCCoR Pilot Program.

Purpose: Only one chemical class of topoisomerase I (TOP1) inhibitors is FDA approved, the camptothecins with irinotecan and topotecan widely used. Because of their limitations (chemical instability, drug efflux-mediated resistance, and diarrhea), novel TOP1 inhibitors are warranted. Indenoisoquinoline non-camptothecin topoisomerase I (TOP1) inhibitors overcome chemical instability and drug resistance that limit camptothecin use. Three indenoisoquinolines, LMP400 (indotecan), LMP776 (indimitecan), and LMP744, were examined in a phase I study for lymphoma-bearing dogs to evaluate differential efficacy, pharmacodynamics, toxicology, and pharmacokinetics. Experimental Design: Eighty-four client-owned dogs with lymphomas were enrolled in dose-escalation cohorts for each indenoisoquinoline, with an expansion phase for LMP744. Efficacy, tolerability, pharmacokinetics, and target engagement were determined. Results: The MTDs were 17.5 mg/m2 for LMP 776 and 100 mg/m2 for LMP744; bone marrow toxicity was dose-limiting; up to 65 mg/m2 LMP400 was well-tolerated and MTD was not reached. None of the drugs induced notable diarrhea. Sustained tumor accumulation was observed for LMP744; γH2AX induction was demonstrated in tumors 2 and 6 hours after treatment; a decrease in TOP1 protein was observed in most lymphoma samples across all compounds and dose levels, which is consistent with the fact that tumor response was also observed at low doses LMP744. Objective responses were documented for all indenoisoquinolines; efficacy (13/19 dogs) was greatest for LMP744. Conclusions: These results demonstrate proof-of-mechanism for indenoisoquinoline TOP1 inhibitors supporting their further clinical development. They also highlight the value of the NCI Comparative Oncology Program (https://ccr.cancer.gov/Comparative-Oncology-Program) for evaluating novel therapies in immunocompetent pets with cancers.

Animal and clinical studies demonstrate that cancer drug combinations (DCs) are more effective than single agents. However, it is difficult to predict which DCs will be more efficacious than individual drugs. Systematic DC high-throughput screening (HTS) of 100 approved drugs in the National Cancer Institute's panel of 60 cancer cell lines (NCI-60) produced data to help select DCs for further consideration. We miniaturized growth inhibition assays into 384-well format, increased the fetal bovine serum amount to 10%, lengthened compound exposure to 72 h, and used a homogeneous detection reagent. We determined the growth inhibition 50% values of individual drugs across 60 cell lines, selected drug concentrations for 4 × 4 DC matrices (DCMs), created DCM master and replica daughter plate sets, implemented the HTS, quality control reviewed the data, and analyzed the results. A total of 2620 DCMs were screened in 60 cancer cell lines to generate 3.04 million data points for the NCI ALMANAC (A Large Matrix of Anti-Neoplastic Agent Combinations) database. We confirmed in vitro a synergistic drug interaction flagged in the DC HTS between the vinca-alkaloid microtubule assembly inhibitor vinorelbine (Navelbine) tartrate and the epidermal growth factor-receptor tyrosine kinase inhibitor gefitinib (Iressa) in the SK-MEL-5 melanoma cell line. Seventy-five percent of the DCs examined in the screen are not currently in the clinical trials database. Selected synergistic drug interactions flagged in the DC HTS described herein were subsequently confirmed by the NCI in vitro, evaluated mechanistically, and were shown to have greater than single-agent efficacy in mouse xenograft human cancer models. Enrollment is open for two clinical trials for DCs that were identified in the DC HTS. The NCI ALMANAC database therefore constitutes a valuable resource for selecting promising DCs for confirmation, mechanistic studies, and clinical translation.

The gastrointestinal (GI) epithelium is the fastest renewing adult tissue and is maintained by tissue-specific stem cells. Treatment-induced GI side effects are a major dose-limiting factor for chemotherapy and abdominal radiotherapy and can decrease the quality of life in cancer patients and survivors. p53 is a key regulator of the DNA damage response, and its activation results in stimulus- and cell type–specific outcomes via distinct effectors. We demonstrate that p53-dependent PUMA induction mediates chemotherapy-induced intestinal injury in mice. Genetic ablation of Puma, but not of p53, protects against chemotherapy-induced lethal GI injury. Blocking chemotherapy- induced loss of LGR5+ stem cells by Puma KO or a small-molecule PUMA inhibitor (PUMAi) prevents perturbation of the stem cell niche, rapid activation of WNT and NOTCH signaling, and stem cell exhaustion during repeated exposures. PUMAi also protects human and mouse colonic organoids against chemotherapy-induced apoptosis and damage but does not protect cancer cells in vitro or in vivo. Therefore, targeting PUMA is a promising strategy for normal intestinal chemoprotection because it selectively blocks p53-dependent stem cell loss but leaves p53-dependent protective effects intact.

// Huabo Wang 1 , Peter Teriete 2 , Angela Hu 1 , Dhanya Raveendra-Panickar 2 , Kelsey Pendelton 1 , John S. Lazo 3 , Julie Eiseman 4,5 , Toril Holien 6 , Kristine Misund 6 , Ganna Oliynyk 7 , Marie Arsenian-Henriksson 7 , Nicholas D. P. Cosford 2 , Anders Sundan 6 and Edward V. Prochownik 1,5,8 1 Section of Hematology/Oncology, Children’s Hospital of Pittsburgh of UPMC, The University of Pittsburgh Medical Center, Pittsburgh, PA, USA 2 Cell Death and Survival Networks Research Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA 3 The Department of Pharmacology, The University of Virginia School of Medicine, Charlottesville, Virginia, USA 4 The Department of Chemical Biology and Pharmacology, The University of Pittsburgh Medical Center, Pittsburgh, PA, USA 5 The University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA 6 Department of Cancer Research and Molecular Medicine and The K. G. Jebsen Center for Myeloma Research, Norwegian University of Science and Technology, Trondheim, Norway 7 Department of Microbiology and Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden 8 The Department of Microbiology and Molecular Genetics, The University of Pittsburgh Medical Center, Pittsburgh, PA, USA Correspondence to: Edward V. Prochownik, email: // Keywords : 10058-F4, 10074-G5, BET inhibitors, myeloma, neuroblastoma, quinone methide Received : September 24, 2015 Accepted : September 26, 2015 Published : October 14, 2015 Abstract Many oncogenic signals originate from abnormal protein-protein interactions that are potential targets for small molecule inhibitors. However, the therapeutic disruption of these interactions has proved elusive. We report here that the naturally-occurring triterpenoid celastrol is an inhibitor of the c-Myc (Myc) oncoprotein, which is over-expressed in many human cancers. Most Myc inhibitors prevent the association between Myc and its obligate heterodimerization partner Max via their respective bHLH-ZIP domains. In contrast, we show that celastrol binds to and alters the quaternary structure of the pre-formed dimer and abrogates its DNA binding. Celastrol contains a reactive quinone methide group that promiscuously forms Michael adducts with numerous target proteins and other free sulfhydryl-containing molecules. Interestingly, triterpenoid derivatives lacking the quinone methide showed enhanced specificity and potency against Myc. As with other Myc inhibitors, these analogs rapidly reduced the abundance of Myc protein and provoked a global energy crisis marked by ATP depletion, neutral lipid accumulation, AMP-activated protein kinase activation, cell cycle arrest and apoptosis. They also inhibited the proliferation of numerous established human cancer cell lines as well as primary myeloma explants that were otherwise resistant to JQ1, a potent indirect Myc inhibitor. N-Myc amplified neuroblastoma cells showed similar responses and, in additional, underwent neuronal differentiation. These studies indicate that certain pharmacologically undesirable properties of celastrol such as Michael adduct formation can be eliminated while increasing selectivity and potency toward Myc and N-Myc. This, together with their low in vivo toxicity, provides a strong rationale for pursuing the development of additional Myc-specific triterpenoid derivatives.

We reported that p62 (sequestosome 1) serves as a signaling hub in bone marrow stromal cells (BMSCs) for the formation of signaling complexes, including NFκB, p38MAPK and JNK, that are involved in the increased osteoclastogenesis and multiple myeloma (MM) cell growth induced by BMSCs that are key contributors to multiple myeloma bone disease (MMBD), and demonstrated that the ZZ domain of p62 (p62-ZZ) is required for BMSC enhancement of MMBD. We recently identified a novel p62-ZZ inhibitor, XRK3F2, which inhibits MM cell growth and BMSC growth enhancement of human MM cells. In the current study, we evaluate the relative specificity of XRK3F2 for p62-ZZ, characterize XRK3F2's capacity to inhibit growth of primary MM cells and human MM cell lines, and test the in vivo effects of XRK3F2 in the immunocompetent 5TGM1 MM model. We found that XRK3F2 induces dramatic cortical bone formation that is restricted to MM containing bones and blocked the effects and upregulation of tumor necrosis factor alpha (TNFα), an osteoblast (OB) differentiation inhibitor that is increased in the MM bone marrow microenvironment and utilizes signaling complexes formed on p62-ZZ, in BMSC. Interestingly, XRK3F2 had no effect on non-MM bearing bone. These results demonstrate that targeting p62 in MM models has profound effects on MMBD.

Purpose: DMS612 is a dimethane sulfonate analog with bifunctional alkylating activity and preferential cytotoxicity to human renal cell carcinoma (RCC) in the NCI-60 cell panel. This first-in-human phase I study aimed to determine dose-limiting toxicity (DLT), maximum tolerated dose (MTD), pharmacokinetics (PK), and pharmacodynamics (PD) of DMS612 administered by 10-minute intravenous infusion on days 1, 8, and 15 of an every-28-day schedule. Experimental Design: Patients with advanced solid malignancies were eligible. Enrollment followed a 3+3 design. PKs of DMS612 and metabolites were assessed by mass spectroscopy and PD by γ-H2AX immunofluorescence. Results: A total of 31 patients, including those with colorectal (11), RCC (4), cervical (2), and urothelial (1) cancers, were enrolled. Six dose levels were studied, from 1.5 mg/m2 to 12 mg/m2. DLTs of grade 4 neutropenia and prolonged grade 3 thrombocytopenia were observed at 12 mg/m2. The MTD was determined to be 9 mg/m2 with a single DLT of grade 4 thrombocytopenia in 1 of 12 patients. Two patients had a confirmed partial response at the 9 mg/m2 dose level, in renal (1) and cervical (1) cancer. DMS612 was rapidly converted into active metabolites. γ-H2AX immunofluorescence revealed dose-dependent DNA damage in both peripheral blood lymphocytes and scalp hairs. Conclusions: The MTD of DMS12 on days 1, 8, and 15 every 28 days was 9 mg/m2. DMS612 appears to be an alkylating agent with unique tissue specificities. Dose-dependent PD signals and two partial responses at the MTD support further evaluation of DMS612 in phase II trials. Clin Cancer Res; 21(4); 721–9. ©2014 AACR.

Bone consists of separate inner endosteal and outer periosteal compartments, each with distinct contributions to bone physiology and each maintaining separate pools of cells owing to physical separation by the bone cortex. The skeletal stem cell that gives rise to endosteal osteoblasts has been extensively studied; however, the identity of periosteal stem cells remains unclear1–5. Here we identify a periosteal stem cell (PSC) that is present in the long bones and calvarium of mice, displays clonal multipotency and self-renewal, and sits at the apex of a differentiation hierarchy. Single-cell and bulk transcriptional profiling show that PSCs display transcriptional signatures that are distinct from those of other skeletal stem cells and mature mesenchymal cells. Whereas other skeletal stem cells form bone via an initial cartilage template using the endochondral pathway4, PSCs form bone via a direct intramembranous route, providing a cellular basis for the divergence between intramembranous versus endochondral developmental pathways. However, there is plasticity in this division, as PSCs acquire endochondral bone formation capacity in response to injury. Genetic blockade of the ability of PSCs to give rise to bone-forming osteoblasts results in selective impairments in cortical bone architecture and defects in fracture healing. A cell analogous to mouse PSCs is present in the human periosteum, raising the possibility that PSCs are attractive targets for drug and cellular therapy for skeletal disorders. The identification of PSCs provides evidence that bone contains multiple pools of stem cells, each with distinct physiologic functions. A periosteal stem cell specialized in intramembranous bone formation has been identified and was found to be essential for normal bone development and fracture healing.

Several cancer chemotherapies cause nausea and vomiting, which can be dose-limiting. Musk shrews are used as preclinical models for chemotherapy-induced emesis and for antiemetic effectiveness. Unlike rats and mice, shrews possess a vomiting reflex and demonstrate an emetic profile similar to humans, including acute and delayed phases. As with most animals, dosing of shrews is based on body weight, while translation of such doses to clinically equivalent exposure requires doses based on body surface area. In the current study body surface area in musk shrews was directly assessed to determine the Meeh constant (Km) conversion factor (female = 9.97, male = 9.10), allowing estimation of body surface area based on body weight. These parameters can be used to determine dosing strategies for shrew studies that model human drug exposures, particularly for investigating the emetic liability of cancer chemotherapeutic agents.

Background The erosion of the early mortality advantage of elective endovascular aneurysm repair (EVAR) compared with open repair of abdominal aortic aneurysm remains without a satisfactory explanation. Methods An individual-patient data meta-analysis of four multicentre randomized trials of EVARversus open repair was conducted to a prespecified analysis plan, reporting on mortality, aneurysm-related mortality and reintervention. Results The analysis included 2783 patients, with 14 245 person-years of follow-up (median 5·5 years). Early (0–6 months after randomization) mortality was lower in the EVAR groups (46 of 1393 versus 73 of 1390 deaths; pooled hazard ratio 0·61, 95 per cent c.i. 0·42 to 0·89; P = 0·010), primarily because 30-day operative mortality was lower in the EVAR groups (16 deaths versus 40 for open repair; pooled odds ratio 0·40, 95 per cent c.i. 0·22 to 0·74). Later (within 3 years) the survival curves converged, remaining converged to 8 years. Beyond 3 years, aneurysm-related mortality was significantly higher in the EVAR groups (19 deaths versus 3 for open repair; pooled hazard ratio 5·16, 1·49 to 17·89; P = 0·010). Patients with moderate renal dysfunction or previous coronary artery disease had no early survival advantage under EVAR. Those with peripheral artery disease had lower mortality under open repair (39 deaths versus 62 for EVAR; P = 0·022) in the period from 6 months to 4 years after randomization. Conclusion The early survival advantage in the EVAR group, and its subsequent erosion, were confirmed. Over 5 years, patients of marginal fitness had no early survival advantage from EVAR compared with open repair. Aneurysm-related mortality and patients with low ankle : brachial pressure index contributed to the erosion of the early survival advantage for the EVAR group. Trial registration numbers: EVAR-1, ISRCTN55703451; DREAM (Dutch Randomized Endovascular Aneurysm Management), NCT00421330; ACE (Anévrysme de l'aorte abdominale, Chirurgie versus Endoprothèse), NCT00224718; OVER (Open Versus Endovascular Repair Trial for Abdominal Aortic Aneurysms), NCT00094575.

Cisplatin induces nausea and emesis, even with antiemetic supportive care. To assess platinum exposure, which could activate nausea and emesis, we quantitated platinum in the brain and various organs, and hindbrain and spinal cord substance P, a key neuropeptide for the neuronal signaling of nausea and emesis.Musk shrews, a model species for nausea and emesis research, were dosed intraperitoneally with 20 mg/kg cisplatin and euthanized at up to 72 h after injection. Concentrations of platinum were quantitated in plasma ultrafiltrate, plasma, lung, kidney, combined forebrain and midbrain, hindbrain, and spinal cord by flameless atomic absorption spectrometry. Hindbrains and spinal cords were analyzed for substance P by immunohistochemistry after injection of 20 or 30 mg/kg.Plasma ultrafilterable platinum concentrations decreased rapidly till 60 min after dosing and then more slowly by 24 h. The concentrations of total platinum in both the fore- and midbrain and the hindbrain were similar at all time points and were at least 20-fold lower than plasma total platinum concentrations. There were no significant changes in substance P immunoreactivity after cisplatin dosing. Histology revealed damage to the renal cortex by 72 h after injection of cisplatin.This is the first study to examine platinum concentrations in musk shrews after administration of cisplatin and delineate substance P immunohistochemical staining in the hindbrain and spinal cord of this species. The platinum concentrations detected in the brain could potentially contribute to the neurological side effects of cisplatin, such as nausea and emesis.

Photodynamic Therapy (PDT) holds great promise for the treatment of head and neck (H&N) carcinomas where repeated loco-regional therapy often becomes necessary due to the highly aggressive and recurrent nature of the cancers. While interstitial light delivery technologies are being refined for PDT of H&N and other cancers, a parallel clinically relevant research area is the formulation of photosensitizers in nanovehicles that allow systemic administration yet preferential enhanced uptake in the tumor. This approach can render dual-selectivity of PDT, by harnessing both the drug and the light delivery within the tumor. To this end, we report on a cell-targeted nanomedicine approach for the photosensitizer silicon phthalocyanine-4 (Pc 4), by packaging it within polymeric micelles that are surface-decorated with GE11-peptides to promote enhanced cell-selective binding and receptor-mediated internalization in EGFR-overexpressing H&N cancer cells. Using fluorescence spectroscopy and confocal microscopy, we demonstrate in vitro that the EGFR-targeted Pc 4-nanoformulation undergoes faster and higher uptake in EGFR-overexpressing H&N SCC-15 cells. We further demonstrate that this enhanced Pc 4 uptake results in significant cell-killing and drastically reduced post-PDT clonogenicity. Building on this in vitro data, we demonstrate that the EGFR-targeted Pc 4-nanoformulation results in significant intra-tumoral drug uptake and subsequent enhanced PDT response, in vivo, in SCC-15 xenografts in mice. Altogether our results show significant promise towards a cell-targeted photodynamic nanomedicine for effective treatment of H&N carcinomas.

Determining the presence and severity of obstructive sleep apnea (OSA) is based on apnea and hypopnea event rates per hour of sleep. Making this determination presents a diagnostic challenge, given that summary metrics do not consider certain factors that influence severity, such as body position and the composition of sleep stages.We retrospectively analyzed 300 consecutive diagnostic PSGs performed at our center to determine the impact of body position and sleep stage on sleep apnea severity.The median percent of REM sleep was 16% (reduced compared to a normal value of ~25%). The median percent supine sleep was 65%. Fewer than half of PSGs contained10 min in each of the 4 possible combinations of REM/NREM and supine/non-supine. Half of patients had2-fold worsening of the apnea-hypopnea index (AHI) in REM sleep, and 60% had2-fold worsening of AHI while supine. Adjusting for body position had greater impact on the AHI than adjusting for reduced REM%. Misclassification--specifically underestimation of OSA severity--is attributed more commonly to body position (20% to 40%) than to sleep stage (~10%).Supine-dominance and REM-dominance commonly contribute to AHI underestimation in single-night PSGs. Misclassification of OSA severity can be mitigated in a patient-specific manner by appropriate consideration of these variables. The results have implications for the interpretation of single-night measurements in clinical practice, especially with trends toward home testing devices that may not measure body position or sleep stage.

Protein kinase D (PKD) mediates diverse biological responses including cell growth and survival. Therefore, PKD inhibitors may have therapeutic potential. We evaluated the in vitro cytotoxicity of two PKD inhibitors, kb-NB142-70 and its methoxy analogue, kb-NB165-09, and examined their in vivo efficacy and pharmacokinetics. The in vitro cytotoxicities of kb-NB142-70 and kb-NB165-09 were evaluated by MTT assay against PC-3, androgen-independent prostate cancer cells, and CFPAC-1 and PANC-1, pancreatic cancer cells. Efficacy studies were conducted in mice bearing either PC-3 or CPFAC-1 xenografts. Tumor-bearing mice were euthanized between 5 and 1,440 min after iv dosing, and plasma and tissue concentrations were measured by HPLC–UV. Metabolites were characterized by LC–MS/MS. kb-NB142-70 and kb-NB165-09 inhibited cellular growth in the low–mid μM range. The compounds were inactive when administered to tumor-bearing mice. In mice treated with kb-NB142-70, the plasma C max was 36.9 nmol/mL, and the PC-3 tumor C max was 11.8 nmol/g. In mice dosed with kb-NB165-09, the plasma C max was 61.9 nmol/mL, while the PANC-1 tumor C max was 8.0 nmol/g. The plasma half-lives of kb-NB142-70 and kb-NB165-09 were 6 and 14 min, respectively. Both compounds underwent oxidation and glucuronidation. kb-NB142-70 and kb-NB165-09 were rapidly metabolized, and concentrations in tumor were lower than those required for in vitro cytotoxicity. Replacement of the phenolic hydroxyl group with a methoxy group increased the plasma half-life of kb-NB165-09 2.3-fold over that of kb-NB142-70. Rapid metabolism in mice suggests that next-generation compounds will require further structural modifications to increase potency and/or metabolic stability.

Sleep fragmentation of any cause is disruptive to the rejuvenating value of sleep. However, methods to quantify sleep architecture remain limited. We have previously shown that human sleep–wake stage distributions exhibit multi-exponential dynamics, which are fragmented by obstructive sleep apnea (OSA), suggesting that Markov models may be a useful method to quantify architecture in health and disease. Sleep stage data were obtained from two subsets of the Sleep Heart Health Study database: control subjects with no medications, no OSA, no medical co-morbidities and no sleepiness (n = 374); and subjects with severe OSA (n = 338). Sleep architecture was simplified into three stages: wake after sleep onset (WASO); non-rapid eye movement (NREM) sleep; and rapid eye movement (REM) sleep. The connectivity and transition rates among eight ‘generator’ states of a first-order continuous-time Markov model were inferred from the observed (‘phenotypic’) distributions: three exponentials each of NREM sleep and WASO; and two exponentials of REM sleep. Ultradian REM cycling was accomplished by imposing time-variation to REM state entry rates. Fragmentation in subjects with severe OSA involved faster transition probabilities as well as additional state transition paths within the model. The Markov models exhibit two important features of human sleep architecture: multi-exponential stage dynamics (accounting for observed bout distributions); and probabilistic transitions (an inherent source of variability). In addition, the model quantifies the fragmentation associated with severe OSA. Markov sleep models may prove important for quantifying sleep disruption to provide objective metrics to correlate with endpoints ranging from sleepiness to cardiovascular morbidity.

Identifying predictors of subjective sleepiness and severity of sleep apnea are important yet challenging goals in sleep medicine. Classification algorithms may provide insights, especially when large data sets are available. We analyzed polysomnography and clinical features available from the Sleep Heart Health Study. The Epworth Sleepiness Scale and the apnea-hypopnea index were the targets of three classifiers: k-nearest neighbor, naive Bayes and support vector machine algorithms. Classification was based on up to 26 features including demographics, polysomnogram, and electrocardiogram (spectrogram). Naive Bayes was best for predicting abnormal Epworth class (0-10 versus 11-24), although prediction was weak: polysomnogram features had 16.7% sensitivity and 88.8% specificity; spectrogram features had 5.3% sensitivity and 96.5% specificity. The support vector machine performed similarly to naive Bayes for predicting sleep apnea class (0-5 versus >5): 59.0% sensitivity and 74.5% specificity using clinical features and 43.4% sensitivity and 83.5% specificity using spectrographic features compared with the naive Bayes classifier, which had 57.5% sensitivity and 73.7% specificity (clinical), and 39.0% sensitivity and 82.7% specificity (spectrogram). Mutual information analysis confirmed the minimal dependency of the Epworth score on any feature, while the apnea-hypopnea index showed modest dependency on body mass index, arousal index, oxygenation and spectrogram features. Apnea classification was modestly accurate, using either clinical or spectrogram features, and showed lower sensitivity and higher specificity than common sleep apnea screening tools. Thus, clinical prediction of sleep apnea may be feasible with easily obtained demographic and electrocardiographic analysis, but the utility of the Epworth is questioned by its minimal relation to clinical, electrocardiographic, or polysomnographic features.

An 18-year-old woman with recurrent embryonal rhabdomyosarcoma underwent a right subtotal exenteration sparing the eyelids and conjunctiva to remove the tumor. A rectus abdominus muscle free flap was secured to the right temporalis muscle. The temporalis muscle was then advanced into the temporal fossa defect and the rectus abdominus flap placed into the right orbital cavity and right maxillary sinus. An ocular conformer was then placed and a lateral tarsorrhaphy was performed. This surgical technique provides rapid socket rehabilitation with good cosmesis and enables the use of a standard ocular prosthesis.

Topoisomerase I (Topo I) is a recognized target for ovarian, lung, and colorectal cancer therapy. The FDA-approved camptothecin (CPT) Topo I inhibitors, topotecan and irinotecan are labile and their effects are rapidly reversible. The indenoisoquinoline topoisomerase I inhibitors, NSC 743400 and NSC 725776, have been developed as a new generation of Topo I inhibitors and are being advanced to clinical evaluation. To support the clinical development of NSC 743400 and NSC 725776, we developed and validated, according to FDA guidelines, LC-MS/MS assays for the sensitive, accurate and precise quantitation of NSC 743400 and NSC 725776 in 0.2 mL human plasma. After ethyl acetate extraction, separation was achieved with a Synergi Polar RP column and a gradient of 0.1% formic acid in acetonitrile:water. NSC 743400 and NSC 725776 eluted at approximately 3 min, and the total run time was 14 min. Detection consisted of electrospray, positive-mode ionization mass spectrometry. Between 3 and 1,000 ng/mL, accuracy was 96.9-108.2% for NSC 743400 and 95.1-106.7% for NSC 725776, and precision was 80% for both analytes, and ion suppression ranged from -46.7 to 5.7%. The use of isotopically labeled internal standards and a wash phase at the end of the run were necessary to achieve adequate assay performance. Protein binding in human plasma as assessed by equilibrium dialysis showed both indenoisoquinolines to be more than 98% protein bound.

The ability to noninvasively measure photosensitizer concentration at target tissues will allow optimization of photodynamic therapy (PDT) and could improve outcome. In this study, we evaluated whether preirradiation tumor phthalocyanine 4 (Pc 4) concentrations, measured noninvasively by the optical pharmacokinetic system (OPS), correlated with tumor response to PDT. Mice bearing human breast cancer xenografts were treated with 2 mg kg ―1 Pc 4 iv only, laser irradiation (150 J cm ―2 ) only, Pc 4 followed by fractionated irradiation or Pc 4 followed by continuous irradiation. Laser irradiation treatment was initiated when the tumor to skin ratio of Pc 4 concentration reached a maximum of 2.1 at 48 h after administration. Pc 4 concentrations in tumor, as well as in Intralipid in vitro, decreased monoexponentially with laser fluence. Pc 4-PDT resulted in significant tumor regression, and tumor response was similar in the groups receiving either fractionated or continuous irradiation treatment after Pc 4. Tumor growth delay following Pc 4-PDT correlated with OPS-measured tumor Pc 4 concentrations at 24 h prior to PDT (R 2 = 0.86). In excised tumors, OPS-measured Pc 4 concentrations were similar to the HPLC-measured concentrations. Thus, OPS measurements of photosensitizer concentrations can be used to assist in the scheduling of Pc 4-PDT.

Thomas N Robinson, Ben EisemanDepartment of Surgery, University of Colorado at Denver Health Sciences Center, Aurora, CO, USAbstract: Delirium is a common but often undiagnosed complication in the elderly following a major operation. Recognizing the presentation of delirium and the criteria to establish the diagnosis of delirium will improve a clinician’s ability to detect this complication. Treating delirium with environmental, supportive, and pharmacologic interventions reduces the incidence and side effects of postoperative delirium. The purpose of this review is to describe the diagnosis and treatment of postoperative delirium.Keywords: postoperative, delirium, geriatric, elderly, review