Monitoring apoptosis in intact cells by high-resolution magic angle spinning 1 H NMR spectroscopy.

Apoptosis maintains an equilibrium between cell proliferation and cell death. Many diseases, including cancer, develop because of defects in apoptosis. A known metabolic marker of apoptosis is a notable increase in 1 H NMR-observable resonances associated with lipids stored in lipid droplets. However, standard one-dimensional NMR experiments allow the quantification of lipid concentration only, without providing information about physical characteristics such as the size of lipid droplets, viscosity of the cytosol, or cytoskeletal rigidity. This additional information can improve monitoring of apoptosis-based cancer treatments in intact cells and provide us with mechanistic insight into why these changes occur. In this paper, we use high-resolution magic angle spinning (HRMAS) 1 H NMR spectroscopy to monitor lipid concentrations and apparent diffusion coefficients of mobile lipid in intact cells treated with the apoptotic agents cisplatin or etoposide. We also use solution-state NMR spectroscopy to study changes in lipid profiles of organic solvent cell extracts. Both NMR techniques show an increase in the concentration of lipids but the relative changes are 10 times larger by HRMAS 1 H NMR spectroscopy. Moreover, the apparent diffusion rates of lipids in apoptotic cells measured by HRMAS 1 H NMR spectroscopy decrease significantly as compared with control cells. Slower diffusion rates of mobile lipids in apoptotic cells correlate well with the formation of larger lipid droplets as observed by microscopy. We also compared the mean lipid droplet displacement values calculated from the two methods. Both methods showed shorter displacements of lipid droplets in apoptotic cells. Our results demonstrate that the NMR-based diffusion experiments on intact cells discriminate between control and apoptotic cells. Apparent diffusion measurements in conjunction with 1 H NMR spectroscopy-derived lipid signals provide a novel means of following apoptosis in intact cells. This method could have potential application in enhancing drug discovery by monitoring drug treatments in vitro, particularly for agents that cause portioning of lipids such as apoptosis.

Discovery of Pyrazolo[1,5-a]pyrimidine B-Cell Lymphoma 6 (BCL6) Binders and Optimization to High Affinity Macrocyclic Inhibitors.

Inhibition of the protein-protein interaction between B-cell lymphoma 6 (BCL6) and corepressors has been implicated as a therapeutic target in diffuse large B-cell lymphoma (DLBCL) cancers and profiling of potent and selective BCL6 inhibitors are critical to test this hypothesis. We identified a pyrazolo[1,5-a]pyrimidine series of BCL6 binders from a fragment screen in parallel with a virtual screen. Using structure-based drug design, binding affinity was increased 100000-fold. This involved displacing crystallographic water, forming new ligand-protein interactions and a macrocyclization to favor the bioactive conformation of the ligands. Optimization for slow off-rate constant kinetics was conducted as well as improving selectivity against an off-target kinase, CK2. Potency in a cellular BCL6 assay was further optimized to afford highly selective probe molecules. Only weak antiproliferative effects were observed across a number of DLBCL lines and a multiple myeloma cell line without a clear relationship to BCL6 potency. As a result, we conclude that the BCL6 hypothesis in DLBCL cancer remains unproven.

Potent and selective bivalent inhibitors of BET bromodomains.

Proteins of the bromodomain and extraterminal (BET) family, in particular bromodomain-containing protein 4 (BRD4), are of great interest as biological targets. BET proteins contain two separate bromodomains, and existing inhibitors bind to them monovalently. Here we describe the discovery and characterization of probe compound biBET, capable of engaging both bromodomains simultaneously in a bivalent, in cis binding mode. The evidence provided here was obtained in a variety of biophysical and cellular experiments. The bivalent binding results in very high cellular potency for BRD4 binding and pharmacological responses such as disruption of BRD4-mediator complex subunit 1 foci with an EC50 of 100 pM. These compounds will be of considerable utility as BET/BRD4 chemical probes. This work illustrates a novel concept in ligand design-simultaneous targeting of two separate domains with a drug-like small molecule-providing precedent for a potentially more effective paradigm for developing ligands for other multi-domain proteins.

Optimization of a Series of Bivalent Triazolopyridazine Based Bromodomain and Extraterminal Inhibitors: The Discovery of (3R)-4-[2-[4-[1-(3-Methoxy-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)-4-piperidyl]phenoxy]ethyl]-1,3-dimethyl-piperazin-2-one (AZD5153).

Here we report the discovery and optimization of a series of bivalent bromodomain and extraterminal inhibitors. Starting with the observation of BRD4 activity of compounds from a previous program, the compounds were optimized for BRD4 potency and physical properties. The optimized compound from this campaign exhibited excellent pharmacokinetic profile and exhibited high potency in vitro and in vivo effecting c-Myc downregulation and tumor growth inhibition in xenograft studies. This compound was selected as the development candidate, AZD5153. The series showed enhanced potency as a result of bivalent binding and a clear correlation between BRD4 activity and cellular potency.

Discovery and optimization of a novel series of Dyrk1B kinase inhibitors to explore a MEK resistance hypothesis.

Potent and selective inhibitors of Dyrk1B kinase were developed to explore the hypothesis, based on siRNA studies, that Dyrk1B may be a resistance mechanism in cells undergoing a stress response.

Symptomatic lumbar osteochondroma treated via a multidisciplinary military surgical team: case report and review of the literature.

The authors describe the case of a giant osteochondroma emanating from the L5 vertebral body and extending into the retroperitoneum of a 40-year-old man, causing low back pain. Osteochondromas are benign bony tumors that typically occur within the appendicular skeleton, although in the sporadic form, up to 4% occur in the spine. A review of the English language literature has returned 44 cases of lumbar osteochondroma, including the present example. The lesions were sporadic in 81% of cases. Mean age of presentation overall is 39.5 years, with a mean age of 18.4 years (range 8-34 years) for hereditary cases and 45.7 years (range 11-81 years) for solitary lesions. Of the instances where gender was reported, 64% were male. The most common level of origin was L4 (38%). The most common anatomic site of origin was the inferior articular process (one-third). Of those lesions treated operatively, 46% underwent simple decompression, with 22% requiring decompression and fusion. This particular lesion was resected via a transperitoneal approach performed by a multidisciplinary team of neurosurgeons, vascular surgeons, and urologists. The bony tumor measured 6.1 × 7.8 × 7.7 cm. Removal of the lesion resulted in a significant improvement of the patient's symptoms.

Intraoperative duplex ultrasound criteria for performing interposition bypass in the treatment of popliteal artery entrapment syndrome.

We present a case series of 3 surgical procedures (2 patients) in which intraoperative duplex ultrasound (IDUS) was used to determine whether the chronic compression of the popliteal artery caused by popliteal artery entrapment syndrome had injured the artery to such a degree that interposition bypass was required. Patients initially underwent standard clinical evaluation including history and physical examination and noninvasive diagnostic testing including postexercise ankle-brachial indexes and angiography with evocative maneuvers before surgery. IDUS was performed. Doppler was used to calculate peak systolic velocities (PSVs) and velocity ratios (VRs) across areas of suspected injury. B-mode was used to assess arterial wall thickness (AWT) and sclerotic changes. Patients were followed in the postoperative period with surveillance duplex ultrasound (US). Three limbs (2 patients) underwent IDUS evaluation after popliteal decompression. Limb 1 demonstrated an elevated intraoperative PSV of 295 cm/sec with an elevated VR of 2.52 (295/117 cm/sec) and AWT of 1.1 mm. Interposition bypass was performed after popliteal decompression. Postoperative surveillance duplex US revealed a reduction of the PSV to 90 cm/sec. Limb 2 showed a mildly elevated intraoperative PSV of 211.5 cm/sec with a VR of 1.86 (211.5/114 cm/sec) and AWT of 0.8 mm. An interposition bypass was not performed. Limb 3 demonstrated an elevated intraoperative PSV of 300 cm/sec with an elevated VR of 2.51 (300/119.5 cm/sec) and AWT of 1.0. Interposition bypass was performed. Postoperative surveillance duplex US revealed a reduction of the PSV to 115 cm/sec. IDUS was very helpful in the operative management and intraoperative decision making process for popliteal artery entrapment. An elevated PSV of 250-275 cm/sec or greater on IDUS and a VR of 2.0 or greater, in conjunction with B-mode demonstration of arterial wall injury, was useful in identifying severely injured popliteal arterial segments. Additional prospective studies are warranted to further investigate objective criteria that indicate the need for bypass.

A noninterventional management approach to endovascular coil migration into the sigmoid colon.

A 75-year-old man underwent endovascular treatment of a right internal iliac artery (IIA) aneurysm by placing coils in the distal IIA and occluding the inflow with a common iliac artery-to-external iliac artery stent graft. Surveillance computed tomography angiography discovered migration of an endovascular coil from the thrombosed right IIA into the sigmoid colon. Subsequent serial imaging demonstrated uncomplicated extracorporeal passage of the coil. We review the relevant literature and treatment rationale.

Tumescent technique without epinephrine for endovenous laser therapy and serum lidocaine concentration.

OBJECTIVE: Endovenous laser therapy (EVLT) requires tumescent lidocaine anesthesia. Although it is well known that the absorption of local anesthetic varies according to the injection site, little evidence exists establishing the maximum recommended safe dose for extravascular injections such as those used for EVLT. The aim of this study was to evaluate plasma concentration of lidocaine over time after administration of tumescent lidocaine during EVLT procedures in healthy volunteers. METHODS: Between January 2011 and February 2013, 10 healthy patients scheduled for an EVLT procedure performed in a hospital setting were recruited to participate in an observational study. For each subject, a total of 10 venous samples were obtained for analysis after surgical injection of the tumescent lidocaine solution (0.1% concentration). Samples were collected at baseline (before the surgical procedure start) and then every 30 minutes for the first 2 hours after the initial lidocaine injection. Thereafter, venous samples were obtained every 2 hours, with the last sample drawn 12 hours after the surgeon's initial lidocaine injection. All specimens were drawn from a dedicated intravenous catheter, immediately placed in a heparinized blood collection tube, and centrifuged for 10 minutes at 3000 rpm. Plasma was then removed with a pipette and stored at -70 °C until analyzed. Total and free plasma lidocaine concentrations were determined by immunoassay. Plasma lidocaine concentrations were normalized by peak concentration for statistical comparisons. RESULTS: Laboratory data were available for nine of the 10 volunteers. The mean total lidocaine dose administered was 6.38 (± 2.2) mg/kg (range, 3.57-10.7 mg/kg). The total lidocaine blood levels ranged from 0.48 (± 0.28) to 1.3 (± 0.49) mcg/mL. The free lidocaine blood levels ranged from nondetectable to 0.76 (± 0.43) mcg/mL. The average total time of injection for the group was 32.8 (± 10.0) minutes (range, 21-49 minutes). Among all dose ranges, both total and free lidocaine peak blood level ratios occurred at times 60 to 120 minutes (P < .05). No value considered in the statistical analysis exceeded 5 mcg/mL at any time. CONCLUSIONS: Tumescent lidocaine without epinephrine for EVLT procedures produces a peak serum concentration at 60 to 120 minutes. The peak plasma concentration as observed with the standard institutional dosing of tumescent lidocaine appeared below the threshold for human toxicity.

Discovery of a potent and selective EGFR inhibitor (AZD9291) of both sensitizing and T790M resistance mutations that spares the wild type form of the receptor.

Epidermal growth factor receptor (EGFR) inhibitors have been used clinically in the treatment of non-small-cell lung cancer (NSCLC) patients harboring sensitizing (or activating) mutations for a number of years. Despite encouraging clinical efficacy with these agents, in many patients resistance develops leading to disease progression. In most cases, this resistance is in the form of the T790M mutation. In addition, EGFR wild type receptor inhibition inherent with these agents can lead to dose limiting toxicities of rash and diarrhea. We describe herein the evolution of an early, mutant selective lead to the clinical candidate AZD9291, an irreversible inhibitor of both EGFR sensitizing (EGFRm+) and T790M resistance mutations with selectivity over the wild type form of the receptor. Following observations of significant tumor inhibition in preclinical models, the clinical candidate was administered clinically to patients with T790M positive EGFR-TKI resistant NSCLC and early efficacy has been observed, accompanied by an encouraging safety profile.

Design and synthesis of novel lactate dehydrogenase A inhibitors by fragment-based lead generation.

Lactate dehydrogenase A (LDHA) catalyzes the conversion of pyruvate to lactate, utilizing NADH as a cofactor. It has been identified as a potential therapeutic target in the area of cancer metabolism. In this manuscript we report our progress using fragment-based lead generation (FBLG), assisted by X-ray crystallography to develop small molecule LDHA inhibitors. Fragment hits were identified through NMR and SPR screening and optimized into lead compounds with nanomolar binding affinities via fragment linking. Also reported is their modification into cellular active compounds suitable for target validation work.

Endovascular coiling of an internal mammary artery pseudoaneurysm following placement of an internal jugular central venous catheter.

We discuss an interesting case of a right internal mammary artery pseudoaneurysm discovered after an attempted right internal jugular venous catheter placement. Such injury to the internal mammary artery is a rare complication of traumatic injuries to the chest, sternotomies, and central venous catheter placements. It has been reported after subclavian line placements, but not after an internal jugular catheterization.

A splice variant of the TATA-box binding protein encoding the polyglutamine-containing N-terminal domain that accumulates in Alzheimer's disease.

Previously we have reported the accumulation of an N-terminal fragment of the TATA-box binding protein (TBP) in Alzheimer's disease brain tissue and here we report the identification of a naturally occurring TBP splice variant as a likely mechanism for its production. The splice variant described here encodes the polyglutamine-containing N-terminal domain of this key transcription factor. We demonstrate the expression of the splice variant mRNA in a variety of human tissues and that the resulting protein forms inclusions in cell culture transfection studies. The unusual properties of the variant protein suggest that it may be functionally relevant in late onset neurodegenerative diseases.

The effect of vein repair on the risk of venous thromboembolic events: a review of more than 100 traumatic military venous injuries.

BACKGROUND: The management of venous trauma remains controversial. Critics of venous repair have cited an increased incidence of associated venous thromboembolic events with this management. We analyzed the current treatment of wartime venous injuries in United States military personnel in an effort to answer this question. METHODS: From December 1, 2001, to October 31, 2005, all United States casualties with named venous injuries were evaluated. A retrospective review of a clinical database was performed on demographics, mechanism of injury, associated injuries, treatment, outcomes, and venous thromboembolic events. Data were analyzed using the Fisher exact test, analysis of variance, and logarithmic transformation. RESULTS: During this 5-year period, 82 patients sustained 103 named venous injuries due to combat operations. All patients were male, with an average age of 27.9 years (range, 20.3-58.3 years). Blast injuries accounted for 54 venous injuries (65.9%), gunshot wounds for 25 (30.5%), and motor vehicle accidents for 3 (3.6%). The venous injury was isolated in 28 patients (34.1%), and 16 (19.5%) had multiple venous injuries. The venous injury in two patients was associated with acute phlegmasia, with fractures in 33 (40.2%), and 22 (28.1%) sustained neurologic deficits. Venous injuries were treated by ligation in 65 patients (63.1%) and by open surgical repair in 38 (36.9%). Postoperative extremity edema occurred in all patients irrespective of method of management. Thrombosis after venous repair occurred in six of the 38 cases (15.8%). Pulmonary emboli developed in three patients, one after open repair and two after ligation (P > .99). CONCLUSION: In the largest review of military venous trauma in more than three decades, we found no difference in the incidence of venous thromboembolic complications between venous injuries managed by open repair vs ligation. Blast injuries of the extremities have caused most of the venous injuries. Ligation is the most common modality of treatment in combat zones. Long-term morbidity associated with venous injuries and their management will be assessed in future follow-up studies.

Traumatic pseudoaneurysms of the head and neck: early endovascular intervention.

BACKGROUND: Trauma to the head and neck with military munitions often presents with complex multisystem injury patterns. Vascular evaluation typically focuses on the carotid and vertebral arteries; however, trauma to branches of the external carotid artery may also result in devastating complications. Pseudoaneurysms are the most frequent finding on delayed evaluation and can result in life-threatening episodes of rebleeding. METHODS: Patients evacuated from the Afghanistan and Iraq conflicts with penetrating injury to the face and neck were evaluated by the vascular surgery service to determine the potential for unsuspected vascular injury. Patients with significant penetrating injury underwent computed tomography angiography (CTA) as the initial evaluation and subsequent arteriography in cases where injuries were suspected or metallic fragments produced artifacts obscuring the vasculature. Data on all vascular evaluations were entered prospectively into a database and retrospectively reviewed. RESULTS: Between February 2003 and March 2007, 124 patients were evaluated for significant penetrating trauma to the head and neck. Thirteen pseudoaneurysms of the head and neck were found in 11 patients: two in the internal carotid artery, one of the vertebral artery, and 10 involving branches of the external carotid. Seven pseudoaneurysms were symptomatic, of which two presented with episodes of massive bleeding and airway compromise. Seven pseudoaneurysms were treated with coil embolization, 1 with Gelfoam (Upjohn, Kalamazoo, Mich) embolization, 2 with stent grafts, 2 with open repair, and 1 with observation alone. None of the patients undergoing embolization had complications; however, a stent graft of the internal carotid artery occluded early, without stroke. All of the pseudoaneurysms had resolved on follow-up CTA or angiogram. CONCLUSIONS: Pseudoaneurysms are a common finding in patients with high-velocity gunshot wounds or blast injuries to the head and neck. Most involve branches of the external carotid artery and can be treated by embolization. CTA should be performed on all patients with high-velocity gunshot wounds or in cases of blast trauma with fragmentation injuries of the head and neck.

The engineering of endovascular stent technology: a review.

The evolution of minimally invasive endovascular technology has initiated a significant paradigm shift in the treatment of vascular disease. A fundamental understanding of the science and engineering behind the technology of endovascular stents is a key to their appropriate implementation in practice. Furthermore, the rapid influx of new devices into the field requires practitioners to make their decisions on a foundation of the relative strengths and weaknesses of the various products. Although the principles of their use are not complex, the device design can have a profound effect on the device's functionality. Shape, thickness, coating, material selection, and imaging are just a few of the factors to consider in stent design. Subtle differences may have profound results. This review is designed to provide the reader with an overview of fundamental concepts that will aide the assessment of new technology.

Shifting Wallgraft position: case reports and review of the forces affecting Wallgraft positioning.

Extensive use of stent-grafts in the iliac arteries is an increasingly common endovascular alternative to an aortobifemoral bypass procedure. The treatment of diffuse bilateral disease is frequently performed by using the technique of paired stent-grafts in the proximal common iliac arteries to effectively re-create the aortic-bifurcation. We present three patients treated with this technique who returned with recurrent occlusive disease. During subsequent treatment, the stent-grafts were noted to be in different positions from where they were initially deployed. A discussion of the possible explanations for the shifting positions follows.

Restenosis in gold-coated renal artery stents.

BACKGROUND: Gold coating improves stent visibility under fluoroscopy. This is particularly valuable for precise stent placement during renal artery stenting (RAS). There is conflicting evidence regarding restenosis with gold-coated stents. To evaluate the effect of gold coating on restenosis after renal stenting, we reviewed the results of all patients undergoing RAS in our practice. METHODS: A retrospective cohort study of all patients undergoing RAS between June 2000 and September 2003 was performed. During this time, both gold-coated and stainless steel stents were used. Restenosis (>60% diameter) was determined by serial follow-up duplex exams (peak systolic velocity >180 cm/s and renal-aortic ratio >3.5). Restenosis rates were determined by using the Kaplan-Meier life-table method. Variables potentially affecting restenosis were evaluated with the log-rank test and Cox proportional hazards modeling. RESULTS: RAS was performed in 97 arteries (78 patients). Gold-coated (NIRoyal) stents were placed in 59 arteries (48 patients). Stainless steel stents (Corinthian, Genesis, and Herculink) were placed in 38 arteries (34 patients). Patient demographics, indication for treatment, technical success, and complications did not differ between gold and stainless steel stent groups. Mean follow-up was 15 months for gold-coated stents and 18 months for stainless steel stents (NS). By life-table method, 1-year and 2-year freedom from restenosis rates were 84% and 78% in arteries treated with stainless steel stents versus 69% and 39% in those treated with gold-coated stents (P = .012, log-rank test). By multivariate analysis, only the use of gold-coated stents (P = .018; hazard ratio [HR], 3.3; 95% confidence interval [CI], 1.2 to 8.7) and bilateral disease (P = .046; HR, 2.3; 95% CI, 1.02 to 5.2) predicted restenosis. Stent diameter, patient demographics, and indication for RAS had no effect on restenosis by univariate analysis. According to American Heart Association criteria, 87% of patients in the stainless steel group had improved blood pressure at 1 year, compared with 77% in the gold-coated stent group (Kaplan-Meier; P = .042, log-rank test). There were no significant differences in the effect of RAS on serum creatinine levels between the two groups. CONCLUSION: Gold-coated renal stents had a substantially higher rate of restenosis than stainless steel stents in our series. These findings have led us to abandon the use of gold-coated stents for RAS. Patients who have received gold-coated stents for the treatment of atherosclerotic renal artery stenosis should be followed closely for evidence of restenosis.