Influence of feline brachycephaly on respiratory, gastrointestinal, sleep, and activity abnormalities.

OBJECTIVE: To determine the influence of brachycephaly on respiratory, gastrointestinal, sleep, and activity-related parameters in cats. STUDY DESIGN: Prospective questionnaire-based study. ANIMALS: A total of 194 BC and 1003 non-BC cats. METHODS: Owners completed an online questionnaire regarding respiratory, gastrointestinal, sleep, and activity-related parameters. Response options were scored, and individual scores summed to give a total clinical severity score for each cat. RESULTS: Brachycephalic cats had more frequent snoring (odds ratio [OR] 6.89; 95% confidence interval [CI]: 5.06-9.41), sneezing (OR 6.52; CI: 4.75-8.98), nasal discharge (OR 8.26; 95% CI 5.77-11.85), coughing (OR 1.75; CI: 1.17-2.59), and dyspnea (OR 5.32; CI: 3.42-8.28); shorter activity before becoming dyspneic (OR 2.71; CI: 1.93-3.79), slower recovery from activity (OR 3.17; CI: 2.19-4.57), lower activity levels (OR 2.16; CI: 1.59-2.95), and increased respiratory noise (OR 6.68; CI: 4.71-9.52); and more hypersalivation (OR 2.50; CI: 1.47-4.16), halitosis (OR 1.40; CI: 1.00-1.95), and difficulty chewing (OR 5.19; CI: 3.65-7.38). Median clinical severity scores were higher for BC cats than non-BC cats (p < .0001). CONCLUSIONS: Brachycephalic cats (BC) were at risk for respiratory, gastrointestinal, and activity-related symptoms compared to non-BC cats. CLINICAL RELEVANCE: Some BC cats exhibit clinically relevant symptoms and behaviors as reported by owners. Medical or surgical interventions may improve these symptoms and warrant investigation.

Ala vestibuloplasty improves cardiopulmonary and activity-related parameters in brachycephalic cats.

OBJECTIVE: To evaluate the effects of ala vestibuloplasty on cardiopulmonary and lifestyle-related parameters in brachycephalic (BC) cats. STUDY DESIGN: Prospective cohort. ANIMALS: Client-owned BC cats (n = 19). METHODS: Cats were assessed preoperatively by airway computed tomography (CT), endoscopy, contrast echocardiography, cardiac biomarkers, and structured owner questionnaire. Ala vestibuloplasty was performed bilaterally, and blood values, imaging, and owner questionnaire responses were re-evaluated 8-20 weeks postoperatively. RESULTS: Cats were presented for predominantly respiratory clinical signs attributable to brachycephaly. Preoperatively, all cats had stenotic nares, prolonged normalized pulmonary transit time (nPTT) (mean 5.43 ± 1.10 s), and a hyperattenuating pulmonary pattern. No complications occurred following surgery. Postoperatively, nPTT (mean 3.89 ± 0.74 s, p < .001) and frequencies of sneezing (p = .002), snoring (p = .006), open-mouth breathing (p = .0004), and nasal discharge (p = .019) were decreased. Cats exhibited increased activity (p = .005), less frequent dyspnea during activity (p < .001), longer duration of activity before becoming dyspneic (p = .002), faster recovery from activity (p < .001), and decreased respiratory noise (p < .001). Median questionnaire scores improved from preoperative to postoperative (p < .001). CONCLUSION: Anatomic, echocardiographic, and CT changes were common in this cohort of clinically affected BC cats. Pulmonary blood flow and respiratory function were improved after surgery. CLINICAL SIGNIFICANCE: Stenotic nares are the predominant airway abnormality in BC cats. Ala vestibuloplasty is a safe procedure that improves cardiac and CT abnormalities and respiratory and other clinical signs in BC cats.

CT volumetric analysis permits comparison of tongue size and tongue fat in different canine brachycephalic and mesaticephalic breeds.

While macroglossia is a newly accepted component of brachycephalic obstructive airway syndrome (BOAS) in dogs, macroglossia with increased tongue fat is a well-known cause for obstructive sleep apnea (OSA) in people, and targeted reduction procedures such as midline glossectomy are used to treat people with OSA. While midline glossectomy has been described in dogs, tissue contributions to macroglossia have not been characterized. The purpose of this retrospective, descriptive, case-control study was to describe and compare volumetric dimensions of the tongue and tongue fat in brachycephalic (BC) and mesaticephalic (MC) dogs using CT images. Data collected included head and neck CT images from 17 BC and 18 control MC dogs. Multiplanar reformatted and 3D reconstructed images were created using image segmentation and specialized visualization software to calculate volumetric dimensions of the total tongue, tongue fat, and tongue muscle. Rostral and caudal topographical distributions of fat were compared. Total tongue and tongue muscle volume (P < 0.0001) and tongue fat volume (P = 0.01) normalized to body weight (BW) were greater in BC dogs. More fat was localized in the caudal tongue in both groups (P < 0.04). In regression analysis, BC conformation and increased weight were significant predictors of increased tongue fat volume. As in people, increased tongue fat may contribute to macroglossia and sleep-disordered breathing in BC dogs. Use of CT volumetry to identify tongue fat deposits may permit targeted surgical reduction of tongue volume in BC dogs and contribute substantially to treatment of BOAS.

Intraoperative Label-Free Multimodal Nonlinear Optical Imaging for Point-of-Procedure Cancer Diagnostics.

Intraoperative imaging in surgical oncology can provide information about the tumor microenvironment as well as information about the tumor margin. Visualizing microstructural features and molecular and functional dynamics may provide important diagnostic and prognostic information, especially when obtained in real-time at the point-of-procedure. A majority of current intraoperative optical techniques are based on the use of the labels, such as fluorescent dyes. However, these exogenous agents disrupt the natural microenvironment, perturb biological processes, and alter the endogenous optical signatures that cells and the microenvironment can provide. Portable nonlinear imaging systems have enabled intraoperative imaging for real-time detection and diagnosis of tissue. We review the development of a label-free multimodal nonlinear optical imaging technique that was adapted into a portable imaging system for intraoperative optical assessment of resected human breast tissue. New developments have applied this technology to assessing needle-biopsy specimens. Needle-biopsy procedures most always precede surgical resection and serve as the first sampling of suspicious masses for diagnosis. We demonstrate the diagnostic feasibility of imaging core needle-biopsy specimens during veterinary cancer surgeries. This intraoperative label-free multimodal nonlinear optical imaging technique can potentially provide a powerful tool to assist in cancer diagnosis at the point-of-procedure.

Influence of normograde versus retrograde catheterization of bile ducts in dogs treated for gallbladder mucocele.

OBJECTIVE: To determine the influence of normograde (NG) versus retrograde (RG) catheterization of the cystic duct and common bile duct (CBD) in dogs with gallbladder mucoceles (GBM) treated with open cholecystectomy. STUDY DESIGN: Retrospective study. ANIMALS: Dogs (n = 117) with GBM. METHODS: Medical records were reviewed for signalment, history, clinical laboratory and diagnostic imaging findings, details of surgery including catheterization method, complications, and outcome. Long-term follow-up data were obtained by telephone or electronic communication. Relationships between catheterization method and clinical variables and outcome were evaluated. RESULTS: Dogs catheterized RG were more likely to experience any postoperative complication (p = .0004) including persistence of gastrointestinal signs (p = .0003). Survival to discharge and long-term survival did not differ by group (p = .23 and p = .49). Total bilirubin (TB) decreased by 70.3% after NG catheterization compared to 39.1% after RG catheterization (p = .03) and increased in 14.9% dogs catheterized NG and 38.0% dogs catheterized RG (p = .004). The presence of a diplomate surgeon at surgery resulted in decreased incidences of any perioperative or postoperative complication (p = .003 and p = .05). CONCLUSION: Retrograde catheterization was associated with more postoperative concerns than NG catheterization, but similar survival times. Surgery should be performed by diplomates experienced in biliary surgery to minimize complications. CLINICAL SIGNIFICANCE: Although both NG and RG techniques to catheterize the cystic duct and CBD are options for treatment of GBM with low mortality, results of this study provide some evidence to recommend NG over RG catheterization.

Biomechanical properties of canine staphylectomies closed with barbed or smooth suture.

OBJECTIVE: To compare the duration of closure and biomechanical properties of staphylectomies closed with absorbable bidirectional barbed suture or smooth monofilament suture in a simple continuous or interrupted pattern STUDY DESIGN: Ex vivo study SAMPLE POPULATION: Soft palates (n = 60) harvested from mesaticephalic canine cadavers METHODS: One centimeter of tissue was excised from the caudal border of each soft palate, and the oral and nasopharyngeal mucosal surfaces were apposed with 2-0 bidirectional Quill Monoderm knotless closure device barbed suture (Q), 3-0 Monocryl in a simple continuous (MC) pattern, or 3-0 Monocryl in a simple interrupted (MI) pattern (n = 20 per group). Duration of closure was compared between groups. Tissues were tested under tension to failure, and mode of failure data were collected by video capture. RESULTS: Closure time was longer for MI closures than for Q and MC closures, with means of 259.9, 215.4, and 196.7 seconds, respectively (P < .0001). No difference was detected in yield force, force to first tissue rupture, maximum force, and energy required for yield and maximum force between groups. Energy to yield was 190.0, 167.8, and 188.95 N-mm for MI, Q, and MC closures, respectively. CONCLUSION: Biomechanical properties of staphylectomies closed with barbed or smooth sutures did not differ in this cadaveric model. CLINICAL SIGNIFICANCE: Barbed suture can be considered as an alternative for closure of canine staphylectomies. These results provide evidence to justify additional research to evaluate clinical outcomes in dogs undergoing staphylectomy.

Preoperative vascular surgery model using a single polymer tough hydrogel with controllable elastic moduli.

Materials used in organ mimics for medial simulation and education require tissue-like softness, toughness, and hydration to give clinicians and students accurate tactile feedback. However, there is a lack of materials that satisfy these requirements. Herein, we demonstrate that a stretchable and tough polyacrylamide hydrogel is useful to build organ mimics that match softness, crack growth resistance, and interstitial water of real organs. Varying the acrylamide concentration between 29 or 62% w/w with a molar ratio between cross-linker and acrylamide of 1 : 10 800 resulted in a fracture energy around ∼2000 J m-2. More interestingly, this tough gel permitted variation of the elastic modulus from 8 to 62 kPa, which matches the softness of brain to vascular and muscle tissue. According to the rheological frequency sweep, the tough polyacrylamide hydrogels had a greatly decreased number of flow units, indicating that when deformed, stress was dispersed over a greater area. We propose that such molecular dissipation results from the increased number of entangled polymers between distant covalent cross-links. The gel was able to undergo various manipulations including stretching, puncture, delivery through a syringe tip, and suturing, thus enabling the use of the gel as a blood vessel model for microsurgery simulation.

Assessment of Emergency Department Antibiotic Discharge Prescription Dosing Errors for Pediatric Patients in a Community Hospital Health System.

OBJECTIVES: We quantify and describe emergency department antibiotic discharge prescription dosing errors for pediatric patients in a community hospital health system. METHODS: This was a retrospective chart review evaluating emergency department discharge prescriptions written between July 1, 2014, and June 30, 2015. Pediatric patients who received a prescription for an oral antibiotic were included in error analysis if they had a weight updated in the electronic medical record during the encounter. We used a predefined threshold of +10% variance from the recommended dose to quantify error. Prescriber, environmental, and antibiotic specific data were also collected to identify variables associated with high incidence of error. RESULTS: Among the 1934 prescriptions included in our error analysis, we detected 776 (40%) dosing errors. Of the prescriptions reviewed, 288 (15%) contained an overdosing error and 488 (25%) contained an underdosing error. There were 208 underdosing errors written for amoxicillin to treat acute otitis media. These errors represented 43% of the total underdosing errors and had a greater magnitude of variance from the recommended dose compared with overall underdosing errors. CONCLUSIONS: Underdosing of amoxicillin in acute otitis media was a dosing error that occurred frequently throughout our community health system. Further research is needed to identify the clinical impact these errors have on pediatric patients.

Pharmacokinetics of platinum and safety evaluation of carboplatin-impregnated calcium sulfate hemihydrate beads after implantation in healthy cats.

OBJECTIVE: To evaluate the pharmacokinetics (PK) of platinum (Pt) and safety of carboplatin-impregnated calcium sulfate hemihydrate (C-I CSH) beads after implantation in healthy cats. STUDY DESIGN: In vivo experimental study. ANIMALS: Six healthy adult cats. METHODS: Three C-I CSH beads were implanted in muscle pockets over the right and left hemithoraces of each cat (~3.9 mg/kg of Pt; 60.4 mg/m2 of calculated carboplatin). Hematology and blood chemistry were tested at baseline and 3, 7, 14, and 21 days postimplantation. Serum was analyzed for Pt at specific times from 1 hour to 21 days. Tissue was obtained for histopathology and analysis of Pt at 3, 7, 14, and 21 days at standardized distances from implantation sites. RESULTS: Platinum was detected in tissues at all times and distances (range, 0.1-4.19 µg/g). Serum Pt increased up to 2.6 hours (3.25 µg/mL) then decreased sharply. Samples containing muscle had higher Pt compared with samples without muscle (P = .004). Mild hypercalcemia was noted in four cats, and mild inflammatory reaction was noted on histopathology of all samples. CONCLUSION: Platinum was released from C-I CSH beads differentially into surrounding tissues over 21 days. Systemic absorption of Pt was minimal, but mild hypercalcemia occurred. CLINICAL SIGNIFICANCE: Implantation was well tolerated by healthy adult cats. Securing beads within muscle may limit Pt diffusion to targeted tissue. Although Pt concentrations did not achieve levels reported to be cytotoxic for feline sarcoma cells in culture, results provide evidence to support evaluation of efficacy in the tumor microenvironment of cats with locally invasive cancers.

Surgical ligation of patent ductus arteriosus in a dorper sheep.

OBJECTIVE: To report the diagnosis and treatment of a companion dorper wether with patent ductus arteriosus (PDA). STUDY DESIGN: Case report. ANIMAL: An 8-month-old dorper wether presented to its primary care veterinarian for a persistent cough and was referred for suspected heart failure on the basis of physical examination and thoracic radiography. A PDA was diagnosed on echocardiography. METHODS: The sheep underwent cardiac catheterization and angiogram to measure pulmonary arterial and right ventricular (RV) pressures, identify the morphology of the PDA, and determine whether an intravascular occlusion of the PDA was feasible. Pulmonary artery pressure was 84/53 mm Hg (mean = 66), and RV pressures were 79/5 mm Hg (mean = 45); these were consistent with pulmonary hypertension. The size and shape of the PDA precluded vascular occlusion. Instead, the PDA was ligate through a left fourth intercostal approach. RESULTS: The sheep improved clinically after surgery. The PDA seemed closed on echocardiogram 3 days after surgery. Measurement of postoperative fractional shortening was consistent with decreased left ventricular systolic function that had resolved according to follow-up echocardiography. CONCLUSION: We report the first known diagnostic evaluation and successful treatment of naturally occurring PDA in a companion sheep. CLINICAL SIGNIFICANCE: For economically valuable small ruminants, radiographs, echocardiography and cardiac catheterization can be used to diagnose and plan surgical treatment of PDAs, with a potential for a good long-term outcome.

Surgical excision of a feline orbital lacrimal gland adenocarcinoma with adjunctive cryotherapy and carboplatin-impregnated bead implantation.

The purpose of this report was to discuss the diagnosis, treatment, and outcome of a cat with an orbital lacrimal gland adenocarcinoma. A 14.5-year-old spayed female domestic shorthair cat was evaluated for a firm swelling at the left dorsotemporal orbital rim. The orbital mass was excised with preservation of the globe, and adjunctive cryotherapy was performed. A definitive diagnosis of lacrimal gland adenocarcinoma was obtained after histopathologic evaluation and histochemical staining with periodic acid-Schiff and mucicarmine. Thirteen months postoperatively, tumor regrowth occurred with a much larger osteolytic lesion, and a second surgery was performed consisting of tumor excision with implantation of carboplatin-impregnated calcium sulfate hemihydrate beads. The cat has remained free of recurrence 11 months after the second surgery (26 months after initial diagnosis and surgery). A feline orbital lacrimal gland adenocarcinoma was successfully managed utilizing globe-preserving surgical excision with adjunctive cryotherapy and subsequent carboplatin-impregnated bead implantation. Orbital lacrimal gland adenocarcinoma in cats may not be as aggressive as other forms of periocular, head, and neck adenocarcinomas.

In vitro chemosensitivity of feline injection site-associated sarcoma cell lines to carboplatin.

OBJECTIVE: To determine the in vitro chemosensitivity of feline injection site-associated sarcoma (FISAS) cells to carboplatin concentrations generated by elution of carboplatin-impregnated calcium sulfate hemihydrate (CI-CSH) beads. STUDY DESIGN: In vitro study. SAMPLE: Five immortalized cell lines from histologically confirmed, primary FISASs. METHODS: For each cell line, one 96-well microplate was used for each time point (24, 48, 72 hours). In each microplate, 3 wells were seeded with ∼7.5 × 103 cells per well for every carboplatin treatment added, ranging from 5 to 450 µM. Microculture plates were incubated for 24, 48, or 72 hours. Drug efficacy was assessed via a bioreductive fluorometric assay. For apoptosis analysis, 3 wells were seeded with ∼5 × 104 cells per well for every carboplatin treatment added, ranging from 5 to 450 µM. Flow cytometry was performed and the relative percentages of viable, apoptotic, and late apoptotic/necrotic cells were reported. All experiments were run in triplicates. RESULTS: Carboplatin exerted dose-dependent and time-dependent effects on FISAS cell viability. The IC50 values were within the range of carboplatin concentrations eluted from CI-CSH beads. CONCLUSION: Elution of carboplatin from CI-CSH beads generate concentrations sufficient to result in 50% growth inhibition of FISAS cells in vitro. Local tumor control might be achieved by implantation of CI-CSH beads immediately following radical or marginal excision of the primary tumor or by implantation without tumor resection.

Validation of a model of feline ureteral obstruction as a tool for teaching microsurgery to veterinary surgeons.

OBJECTIVE: To assess the content and face validity of a model of an obstructed feline ureter as a tool for teaching ureteral microsurgery. STUDY DESIGN: Prospective, experimental study. SAMPLE POPULATION: Seven expert and 11 novice microsurgeons. METHODS: The model was created from latex rubber with an inner diameter of 0.8 mm and an outer diameter of 5 mm. The "ureter" was created with an inner compartment, a thin wall, and a soft, outer layer mimicking periureteral fat. A "ureterolith" 0.8-1.2 mm in diameter was placed inside the inner compartment by using a blunt-tipped cannula. A standardized "ureterotomy" was performed by 7 expert and 11 novice microsurgeons. Both groups completed questionnaires evaluating the content and face validity of the model using subjective measures and a 5-point Likert scale. Reliability was analysed by calculation of Cronbach's α for all questions to ensure α ≥ .7. The median responses to each question were compared between groups with a nonparametric independent samples median test. P < .05 was considered significant. RESULTS: The Cronbach's α for the experts and the novices for content validity questions was .7 and .9, respectively, and for the face validity questions it was .7 and .8, respectively. The model was rated to have excellent content validity and very good face validity. CONCLUSION AND IMPACT: The model elicited positive responses from expert and novice microsurgeons and can be recommended as a tool for teaching ureteral microsurgery. A model validated by face and content measures should next be scrutinized by determination of construct, concurrent, and predictive validity by using objective measures.

Trastuzumab uptake and its relation to efficacy in an animal model of HER2-positive breast cancer brain metastasis.

PURPOSE: The extent to which efficacy of the HER2 antibody Trastuzumab in brain metastases is limited by access of antibody to brain lesions remains a question of significant clinical importance. We investigated the uptake and distribution of trastuzumab in brain and mammary fat pad grafts of HER2-positive breast cancer to evaluate the relationship of these parameters to the anti-tumor activity of trastuzumab and trastuzumab emtansine (T-DM1). METHODS: Mouse transgenic breast tumor cells expressing human HER2 (Fo2-1282 or Fo5) were used to establish intracranial and orthotopic tumors. Tumor uptake and tissue distribution of systemically administered 89Zr-trastuzumab or muMAb 4D5 (murine parent of trastuzumab) were measured by PET and ELISA. Efficacy of muMAb 4D5, the PI3K/mTOR inhibitor GNE-317, and T-DM1 was also assessed. RESULTS: 89Zr-trastuzumab and muMAb 4D5 exhibited robust uptake into Fo2-1282 brain tumors, but not normal brains. Uptake into brain grafts was similar to mammary grafts. Despite this, muMAb 4D5 was less efficacious in brain grafts. Co-administration of muMAb 4D5 and GNE-317, a brain-penetrant PI3K/mTOR inhibitor, provided longer survival in mice with brain lesions than either agent alone. Moreover, T-DM1 increased survival in the Fo5 brain metastasis model. CONCLUSIONS: In models of HER2-positive breast cancer brain metastasis, trastuzumab efficacy does not appear to be limited by access to intracranial tumors. Anti-tumor activity improved with the addition of a brain-penetrant PI3K/mTOR inhibitor, suggesting that combining targeted therapies is a more effective strategy for treating HER2-positive breast cancer brain metastases. Survival was also extended in mice with Fo5 brain lesions treated with T-DM1.

Hominoid-specific enzyme GLUD2 promotes growth of IDH1R132H glioma.

Somatic mutation of isocitrate dehydrogenase 1 (IDH1) is now recognized as the most common initiating event for secondary glioblastoma, a brain tumor type arising with high frequency in the frontal lobe. A puzzling feature of IDH1 mutation is the selective manifestation of glioma as the only neoplasm frequently associated with early postzygotic occurrence of this genomic alteration. We report here that IDH1(R132H) exhibits a growth-inhibitory effect that is abrogated in the presence of glutamate dehydrogenase 2 (GLUD2), a hominoid-specific enzyme purportedly optimized to facilitate glutamate turnover in human forebrain. Using murine glioma progenitor cells, we demonstrate that IDH1(R132H) exerts a growth-inhibitory effect that is paralleled by deficiency in metabolic flux from glucose and glutamine to lipids. Examining human gliomas, we find that glutamate dehydrogenase 1 (GLUD1) and GLUD2 are overexpressed in IDH1-mutant tumors and that orthotopic growth of an IDH1-mutant glioma line is inhibited by knockdown of GLUD1/2. Strikingly, introduction of GLUD2 into murine glioma progenitor cells reverses deleterious effects of IDH1 mutation on metabolic flux and tumor growth. Further, we report that glutamate, a substrate of GLUD2 and a neurotransmitter abundant in mammalian neocortex, can support growth of glioma progenitor cells irrespective of IDH1 mutation status. These findings suggest that specialization of human neocortex for high glutamate neurotransmitter flux creates a metabolic niche conducive to growth of IDH1 mutant tumors.

Brain Distribution and Efficacy of the Brain Penetrant PI3K Inhibitor GDC-0084 in Orthotopic Mouse Models of Human Glioblastoma.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. Limited treatment options have only marginally impacted patient survival over the past decades. The phophatidylinositol 3-kinase (PI3K) pathway, frequently altered in GBM, represents a potential target for the treatment of this glioma. 5-(6,6-Dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[4,3-e]purin-2-yl)pyrimidin-2-amine (GDC-0084) is a PI3K inhibitor that was specifically optimized to cross the blood-brain barrier. The goals of our studies were to characterize the brain distribution, pharmacodynamic (PD) effect, and efficacy of GDC-0084 in orthotopic xenograft models of GBM. GDC-0084 was tested in vitro to assess its sensitivity to the efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) and in vivo in mice to evaluate its effects on the PI3K pathway in intact brain. Mice bearing U87 or GS2 intracranial tumors were treated with GDC-0084 to assess its brain distribution by matrix-assisted laser desorption ionization (MALDI) imaging and measure its PD effects and efficacy in GBM orthotopic models. Studies in transfected cells indicated that GDC-0084 was not a substrate of P-gp or BCRP. GDC-0084 markedly inhibited the PI3K pathway in mouse brain, causing up to 90% suppression of the pAkt signal. MALDI imaging showed GDC-0084 distributed evenly in brain and intracranial U87 and GS2 tumors. GDC-0084 achieved significant tumor growth inhibition of 70% and 40% against the U87 and GS2 orthotopic models, respectively. GDC-0084 distribution throughout the brain and intracranial tumors led to potent inhibition of the PI3K pathway. Its efficacy in orthotopic models of GBM suggests that it could be effective in the treatment of GBM. GDC-0084 is currently in phase I clinical trials.

Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti-tumor activity as a therapeutic agent.

Binding of hepatocyte growth factor (HGF) to the receptor tyrosine kinase MET is implicated in the malignant process of multiple cancers, making disruption of this interaction a promising therapeutic strategy. However, targeting MET with bivalent antibodies can mimic HGF agonism via receptor dimerization. To address this limitation, we have developed onartuzumab, an Escherichia coli-derived, humanized, and affinity-matured monovalent monoclonal antibody against MET, generated using the knob-into-hole technology that enables the antibody to engage the receptor in a one-to-one fashion. Onartuzumab potently inhibits HGF binding and receptor phosphorylation and signaling and has antibody-like pharmacokinetics and antitumor activity. Biochemical data and a crystal structure of a ternary complex of onartuzumab antigen-binding fragment bound to a MET extracellular domain fragment, consisting of the MET Sema domain fused to the adjacent Plexins, Semaphorins, Integrins domain (MET Sema-PSI), and the HGF ß-chain demonstrate that onartuzumab acts specifically by blocking HGF α-chain (but not ß-chain) binding to MET. These data suggest a likely binding site of the HGF α-chain on MET, which when dimerized leads to MET signaling. Onartuzumab, therefore, represents the founding member of a class of therapeutic monovalent antibodies that overcomes limitations of antibody bivalency for targets impacted by antibody crosslinking.

Elution of Clindamycin and Enrofloxacin From Calcium Sulfate Hemihydrate Beads In Vitro.

OBJECTIVE: To compare the in vitro elution characteristics of clindamycin and enrofloxacin from calcium sulfate hemihydrate beads containing a single antibiotic, both antibiotics, and each antibiotic incubated in the same eluent well. STUDY DESIGN: Experimental in vitro study. METHODS: Calcium sulfate hemihydrate beads were formed by mixing with clindamycin and/or enrofloxacin to create 4 study groups: (1) 160 mg clindamycin/10 beads; (2) 160 mg enrofloxacin/10 beads; (3) 160 mg clindamycin + 160 mg enrofloxacin/10 beads; and (4) 160 mg clindamycin/5 beads and 160 mg enrofloxacin/5 beads. Chains of beads were formed in triplicate and placed in 5 mL phosphate buffered saline (PBS; pH 7.4 and room temperature) with constant agitation. Antibiotic-conditioned PBS was sampled at 14 time points from 1 hour to 30 days. Clindamycin and enrofloxacin concentrations in PBS were determined using high-performance liquid chromatography. RESULTS: Eluent concentrations from clindamycin-impregnated beads failed to remain sufficiently above minimum inhibitory concentration (MIC) for common infecting bacteria over the study period. Enrofloxacin eluent concentrations remained sufficiently above MIC for common wound pathogens of dogs and cats and demonstrated an atypical biphasic release pattern. No significant differences in elution occurred as a result of copolymerization of the antibiotics into a single bead or from individual beads co-eluting in the same eluent well. CONCLUSION: Clindamycin-impregnated beads cannot be recommended for treatment of infection at the studied doses; however, use of enrofloxacin-impregnated beads may be justified when susceptible bacteria are cultured.

NFKBIA deletion in glioblastomas.

BACKGROUND: Amplification and activating mutations of the epidermal growth factor receptor (EGFR) oncogene are molecular hallmarks of glioblastomas. We hypothesized that deletion of NFKBIA (encoding nuclear factor of κ-light polypeptide gene enhancer in B-cells inhibitor-α), an inhibitor of the EGFR-signaling pathway, promotes tumorigenesis in glioblastomas that do not have alterations of EGFR. METHODS: We analyzed 790 human glioblastomas for deletions, mutations, or expression of NFKBIA and EGFR. We studied the tumor-suppressor activity of NFKBIA in tumor-cell culture. We compared the molecular results with the outcome of glioblastoma in 570 affected persons. RESULTS: NFKBIA is often deleted but not mutated in glioblastomas; most deletions occur in nonclassical subtypes of the disease. Deletion of NFKBIA and amplification of EGFR show a pattern of mutual exclusivity. Restoration of the expression of NFKBIA attenuated the malignant phenotype and increased the vulnerability to chemotherapy of cells cultured from tumors with NFKBIA deletion; it also reduced the viability of cells with EGFR amplification but not of cells with normal gene dosages of both NFKBIA and EGFR. Deletion and low expression of NFKBIA were associated with unfavorable outcomes. Patients who had tumors with NFKBIA deletion had outcomes that were similar to those in patients with tumors harboring EGFR amplification. These outcomes were poor as compared with the outcomes in patients with tumors that had normal gene dosages of NFKBIA and EGFR. A two-gene model that was based on expression of NFKBIA and O(6)-methylguanine DNA methyltransferase was strongly associated with the clinical course of the disease. CONCLUSIONS: Deletion of NFKBIA has an effect that is similar to the effect of EGFR amplification in the pathogenesis of glioblastoma and is associated with comparatively short survival.

Distribution of the phosphatidylinositol 3-kinase inhibitors Pictilisib (GDC-0941) and GNE-317 in U87 and GS2 intracranial glioblastoma models-assessment by matrix-assisted laser desorption ionization imaging.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, and the limited available treatment options have not meaningfully impacted patient survival in the past decades. Such poor outcomes can be at least partly attributed to the inability of most drugs tested to cross the blood-brain barrier and reach all areas of the glioma. The objectives of these studies were to visualize and compare by matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry the brain and tumor distribution of the phosphatidylinositol 3-kinase (PI3K) inhibitors pictilisib (GDC-0941, 2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine) and GNE-317 [5-(6-(3-methoxyoxetan-3-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-2-yl)pyrimidin-2-amine] in U87 and GS2 orthotopic models of GBM, models that exhibit differing blood-brain barrier characteristics. Following administration to tumor-bearing mice, pictilisib was readily detected within tumors of the contrast-enhancing U87 model whereas it was not located in tumors of the nonenhancing GS2 model. In both GBM models, pictilisib was not detected in the healthy brain. In contrast, GNE-317 was uniformly distributed throughout the brain in the U87 and GS2 models. MALDI imaging revealed also that the pictilisib signal varied regionally by up to 6-fold within the U87 tumors whereas GNE-317 intratumor levels were more homogeneous. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analyses of the nontumored half of the brain showed pictilisib had brain-to-plasma ratios lower than 0.03 whereas they were greater than 1 for GNE-317, in agreement with their brain penetration properties. These results in orthotopic models representing either the contrast-enhancing or invasive areas of GBM clearly demonstrate the need for whole-brain distribution to potentially achieve long-term efficacy in GBM.