Innovative, combinatorial and high-throughput approaches to degrader synthesis.

Targeted protein degraders such as PROTACs and molecular glues are a rapidly emerging therapeutic modality within industry and academia. Degraders possess unique mechanisms of action that lead to the removal of specific proteins by co-opting the cell's natural degradation mechanisms via induced proximity. Their optimisation thus far has often been largely empirical, requiring the synthesis and screening of a large number of analogues. In addition, the synthesis and development of degraders is often challenging, leading to lengthy optimisation campaigns to deliver candidate-quality compounds. This review highlights how the synthesis of degraders has evolved in recent years, in particular focusing on means of applying high-throughput chemistry and screening approaches to expedite these timelines, which we anticipate to be valuable in shaping the future of degrader optimisation campaigns.

The radial forearm snake flap: An underutilized technique for fasciocutaneous and osteocutaneous forearm flaps with primary closure.

BACKGROUND: The radial forearm free flap (RFFF) is associated with troublesome donor site morbidity related to split thickness skin grafting (STSG). The radial forearm snake flap with primary closure of the donor site may reduce donor site complications. METHODS: Single institution, retrospective cohort study comparing rates of delayed donor site wound healing and tendon exposure in 52 patients undergoing radial forearm snake flap and 95 patients undergoing conventional RFFF with STSG closure of the donor site. RESULTS: Tendon exposure occurred in zero (0%) patients undergoing snake flap and four (4.2%) patients undergoing conventional RFFF (0/52 vs. 4/95; p = 0.297). Delayed wound healing occurred in zero (0%) patients undergoing snake flap and 19 (20.0%) patients undergoing conventional RFFF (0/52 vs. 19/95; p < 0.001). CONCLUSIONS: The radial forearm snake flap provides an alternative to conventional RFFF harvest, which enables primary donor site closure with reduced rates of delayed donor site healing.

siRNA targeted to p53 attenuates ischemic and cisplatin-induced acute kidney injury.

Proximal tubule cells (PTCs), which are the primary site of kidney injury associated with ischemia or nephrotoxicity, are the site of oligonucleotide reabsorption within the kidney. We exploited this property to test the efficacy of siRNA targeted to p53, a pivotal protein in the apoptotic pathway, to prevent kidney injury. Naked synthetic siRNA to p53 injected intravenously 4 h after ischemic injury maximally protected both PTCs and kidney function. PTCs were the primary site for siRNA uptake within the kidney and body. Following glomerular filtration, endocytic uptake of Cy3-siRNA by PTCs was rapid and extensive, and significantly reduced ischemia-induced p53 upregulation. The duration of the siRNA effect in PTCs was 24 to 48 h, determined by levels of p53 mRNA and protein expression. Both Cy3 fluorescence and in situ hybridization of siRNA corroborated a short t(1/2) for siRNA. The extent of renoprotection, decrease in cellular p53 and attenuation of p53-mediated apoptosis by siRNA were dose- and time-dependent. Analysis of renal histology and apoptosis revealed improved injury scores in both cortical and corticomedullary regions. siRNA to p53 was also effective in a model of cisplatin-induced kidney injury. Taken together, these data indicate that rapid delivery of siRNA to proximal tubule cells follows intravenous administration. Targeting siRNA to p53 leads to a dose-dependent attenuation of apoptotic signaling, suggesting potential therapeutic benefit for ischemic and nephrotoxic kidney injury.

Auriculotemporal Nerve Involvement in Parotid Bed Malignancy.

OBJECTIVE: To identify and evaluate patients with parotid bed malignancy demonstrating radiographic findings of auriculotemporal (AT) nerve involvement. METHODS: A retrospective review of patients with parotid bed malignancy was performed to identify patients with imaging findings of AT nerve involvement and record associated clinical findings, symptoms, and pathology information. Independent, blinded review of radiographic images by a senior neuroradiologist was performed to identify imaging characteristics and categorize patients into highly likely or possible involvement groups. RESULTS: Of 547 patients identified with parotid bed malignancy, 23 patients exhibited radiographic findings suggestive of AT nerve involvement. Thirteen patients met criteria for highly likely involvement, and 10 patients met criteria for possible involvement. Cutaneous malignancy with metastasis to the parotid bed accounted for 11 of 23 patients, and the most common histology was squamous cell carcinoma (9 patients). Primary parotid malignancy accounted for 12 of 23 patients, and the most common histology was salivary ductal carcinoma (3 patients). All 13 highly likely patients reported periauricular pain, and 11 of 13 demonstrated facial weakness. Features suggesting advanced disease included radiographic findings of intracranial involvement (10/23 patients), nonsurgical primary treatment (13/23 patients), and positive margins on pathology report (7/10 patients). CONCLUSION: AT nerve involvement is an uncommon but important phenomenon that often occurs in the setting of advanced disease and is commonly associated with periauricular pain and coexisting facial weakness. Awareness of the associated clinical features and imaging patterns can allow for appropriate identification of this pattern of spread and help to optimize treatment planning.

Intradermal delivery of modified mRNA encoding VEGF-A in patients with type 2 diabetes.

Chemically modified mRNA is an efficient, biocompatible modality for therapeutic protein expression. We report a first-time-in-human study of this modality, aiming to evaluate safety and potential therapeutic effects. Men with type 2 diabetes mellitus (T2DM) received intradermal injections of modified mRNA encoding vascular endothelial growth factor A (VEGF-A) or buffered saline placebo (ethical obligations precluded use of a non-translatable mRNA control) at randomized sites on the forearm. The only causally treatment-related adverse events were mild injection-site reactions. Skin microdialysis revealed elevated VEGF-A protein levels at mRNA-treated sites versus placebo-treated sites from about 4-24 hours post-administration. Enhancements in basal skin blood flow at 4 hours and 7 days post-administration were detected using laser Doppler fluximetry and imaging. Intradermal VEGF-A mRNA was well tolerated and led to local functional VEGF-A protein expression and transient skin blood flow enhancement in men with T2DM. VEGF-A mRNA may have therapeutic potential for regenerative angiogenesis.

Profile of a Highly Selective Quaternized Pyrrolidine Betaine αvß6 Integrin Inhibitor-(3S)-3-(3-(3,5-Dimethyl-1H-pyrazol-1-yl)phenyl)-4-((1S and 1R,3R)-1-methyl-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-ium-1-yl)butanoate Synthesized by Stereoselective Methylation.

A quaternary ammonium betaine 7 is described which shows exceptional potency and selectivity (1.4 to >3 logs) for the αvß6 integrin receptor over the other αv integrins as determined in cell adhesion assays. 7 is prepared by remarkably stereoselective methylation, the origins of which are discussed. The chemical, biological, physicochemical, and pharmacokinetic properties of 7 and its docking into αvß6 are described along with related analogues.

Is HPV-Associated Oropharyngeal Cancer Becoming More Common in Older Patients?

OBJECTIVE: To evaluate changing age demographics over a 15-year period for patients with HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). STUDY DESIGN: Retrospective review of patients identified with p16-positive OPSCC at our institution over a 15-year timeframe. Materials/Methods: p16-positive immunohistochemistry was used as a surrogate for HPV-associated OPSCC. Patients were categorized according to year of diagnosis (2002-2010 versus 2011-2016). Mean age and proportion of patients over age 65 were statistically evaluated and compared. RESULTS: From 2002 to 2010, 100 patients were identified with p16-positive OPSCC, mean age at diagnosis was 55.2, and the proportion of patients over 65 was 10.0%. From 2011 to 2016, 188 patients were identified with p16-positive OPSCC, mean age was 58.5, and the proportion of patients over 65 was 19.6%. Both the mean age difference and the difference in proportion of patients over 65 were statistically significant (P = .001 and P = .034, respectively). CONCLUSION: The mean age at diagnosis and proportion of patients over 65 has increased over the past 15 years at our institution. This data suggests that HPV-associated OPSCC is being diagnosed more frequently in older persons and that the age demographic may be shifting. Confirmation of this trend with larger patient numbers on a national level will be valuable. This study highlights the importance of maintaining a high clinical suspicion for HPV-associated OPSCC regardless of patient age. LEVEL OF EVIDENCE: 4.

Excised larynx evaluation of subthyroid cartilage approach to medialization thyroplasty.

OBJECTIVES/HYPOTHESIS: To describe an alternative approach to medialization thyroplasty involving dissection underneath the thyroid cartilage with placement of a Gore-Tex implant, and to evaluate its effect on a range of phonatory measures using an excised canine larynx model. STUDY DESIGN: Animal model. METHODS: On each of eight excised canine larynges, the conditions of normal, paralysis, medialization thyroplasty by standard transthyroid cartilage approach, and medialization thyroplasty by experimental subthyroid cartilage approach were performed. Aerodynamic, acoustic, and mucosal wave parameters were measured for each condition. RESULTS: Compared to the vocal fold paralysis state, both the transthyroid and subthyroid approaches for Gore-Tex insertion resulted in significant decreases in phonation threshold pressure and phonation threshold flow. Both approaches also significantly decreased percent jitter, decreased percent shimmer, and improved signal-to-noise ratio. The mucosal wave was preserved after insertion of the Gore-Tex implant for both approaches. For all the phonatory measures except phonation threshold flow, there were no significant differences between the transthyroid and subthyroid approaches. CONCLUSIONS: Gore-Tex implantation via a subthyroid approach in an excised canine larynx model can produce effective medialization, preserve the mucosal wave, and significantly improve aerodynamic and acoustic parameters without meaningful difference compared to a traditional transthyroid approach. The subthyroid approach does not require creation of a thyroid cartilage window and could be a potentially valuable alternative method of performing medialization thyroplasty. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:675-681, 2018.

Applications of antisense and siRNAs during preclinical drug development.

A significantly greater number of candidate drug targets and compounds are now being generated during preclinical drug development. To date, however, such increases have not led to improvements in clinical success rates or reduced times to market. There is a need for better strategies to prioritize targets and drug candidates. Antisense and siRNA technologies offer exceptional speed and specificity to address this need. In particular, antisense and siRNAs are beginning to be used in combination with expression profiling to evaluate drug specificity and mechanism-of-action, aiding in the identification of better candidates earlier in the drug development process.

Toxicological and pharmacokinetic properties of QPI-1007, a chemically modified synthetic siRNA targeting caspase 2 mRNA, following intravitreal injection.

We report the toxicological and pharmacokinetic properties of the synthetic, small interfering RNA (siRNA), QPI-1007, following intravitreal administration. QPI-1007 is a chemically modified siRNA designed to act via the RNA interference (RNAi) pathway to temporarily inhibit expression of the caspase 2 protein and is being developed as a neuroprotectant for the treatment of nonarteritic anterior ischemic optic neuropathy and other optic neuropathies such as glaucoma that result in the death of retinal ganglion cells. The half-life of QPI-1007 in the vitreous and retina/choroid in the Dutch Belted rabbit was about 2 days, and there was no sign of accumulation after repeated administrations at either 2- or 4-week dosing intervals in the rabbit. QPI-1007 was well tolerated in Dutch Belted rabbits following single or repeated intravitreal administrations of up to 11 doses over 9 months. Test-article-related effects were limited to the eyes, with minimal to mild vitreal cellular infiltration being the major finding, which was reversible. In repeated-dose studies, a modest reduction in B-wave amplitude obtained by electroretinography was observed in animals treated with the highest dose level tested (3 mg, which is equivalent to a 12 mg/eye human dose) that was not considered to be clinically meaningful. Administration in the rat of either a single bolus intravenous (i.v.) injection of 100 mg/kg or daily bolus i.v. injections of 75 mg/kg/day for 28 days failed to elicit any macroscopic or microscopic changes, suggesting a low risk for systemic toxicity. QPI-1007 was negative in three genetic toxicity studies. Overall, the nonclinical studies support the further development of QPI-1007.

Toxicological and pharmacokinetic properties of chemically modified siRNAs targeting p53 RNA following intravenous administration.

We report the toxicological and pharmacokinetic properties of the synthetic, small interfering RNA I5NP following intravenous administration in rodents and nonhuman primates. I5NP is designed to act via the RNA interference (RNAi) pathway to temporarily inhibit expression of the pro-apoptotic protein p53 and is being developed to protect cells from acute ischemia/reperfusion injuries such as acute kidney injury that can occur during major cardiac surgery and delayed graft function that can occur following renal transplantation. Following intravenous administration, I5NP was very rapidly cleared from plasma was distributed predominantly to the kidney, with very low levels in liver and other tissues. Doses of 800 mg/kg I5NP in rodents, and 1,000 mg/kg I5NP in nonhuman primates, were required to elicit adverse effects, which in the monkey were isolated to direct effects on the blood that included a sub-clinical activation of complement and slightly increased clotting times. In the rat, no additional adverse effects were observed with a rat analogue of I5NP, indicating that the effects likely represent class effects of synthetic RNA duplexes rather than toxicity related to the intended pharmacologic activity of I5NP. Taken together, these data support clinical testing of intravenous administration of I5NP for the preservation of renal function following acute ischemia/reperfusion injury.

Gunnerales are sister to other core eudicots: implications for the evolution of pentamery.

Phylogenetic relationships among many lineages of angiosperms have been clarified via the analysis of large molecular data sets. However, with a data set of three genes (18S rDNA, rbcL, and atpB), relationships among lineages of core eudicots (Berberidopsidales, Caryophyllales, Gunnerales, Santalales, Saxifragales, asterids, rosids) remain essentially unresolved. We added 26S rDNA sequences to a three-gene matrix for 201 eudicots (8430 base pair aligned nucleotides per taxon). Parsimony analyses provided moderate (84%) jackknife support for Gunnerales, which comprise the two enigmatic families Gunneraceae and Myrothamnaceae, as sister to all other core eudicots. This position of Gunnerales has important implications for floral evolution. A dimerous or trimerous perianth is frequently encountered in early-diverging eudicots (e.g., Buxaceae, Proteales, Ranunculales, Trochodendraceae), whereas in core eudicots, pentamery predominates. Significantly, dimery is found in Gunneraceae and perhaps Myrothamnaceae (the merosity of the latter has also been interpreted as labile). Parsimony reconstructions of perianth merosity demonstrate lability among early-diverging eudicots and further indicate that a dimerous perianth could be the immediate precursor to the pentamerous condition characteristic of core eudicots. Thus, the developmental canalization that yielded the pentamerous condition of core eudicots occurred after the node leading to Gunnerales.