E3 ubiquitin ligase UBR5 promotes gemcitabine resistance in pancreatic cancer by inducing O-GlcNAcylation-mediated EMT via destabilization of OGA.

Pancreatic cancer (PC) is among the deadliest malignancies, with an extremely poor diagnosis and prognosis. Gemcitabine (GEM) remains the first-line drug for treating PC; however, only a small percentage of patients benefit from current immunotherapies or targeted therapies. Resistance to GEM is prevalent and affects long-term survival. We found that ubiquitin-protein ligase E3 module N-recognition 5 (UBR5) is a therapeutic target against GEM resistance. UBR5 was markedly upregulated in clinical GEM-resistant PC samples and GEM-resistant PC cells. UBR5 knockdown markedly increased GEM sensitivity in GEM-resistant PC cell lines. UBR5-mediated GEM resistance was accompanied by activation of epithelial-mesenchymal transition (EMT) and could be mitigated by inhibiting EMT. Further analysis revealed that UBR5 promoted GEM resistance in PC cells by enhancing O-GlcNAcylation-mediated EMT. In addition, UBR5 knockdown resulted in increased O-GlcNAase (OGA) levels, an essential negatively regulated enzyme in the O-GlcNAcylation process. We identified a negative association between OGA and UBR5 levels, which further supported the hypothesis that O-GlcNAcylation-mediated GEM resistance induced by UBR5 is OGA-dependent in PC cells. Mechanistic studies revealed that UBR5 acts as an E3 ubiquitin ligase of OGA and regulates O-GlcNAcylation by binding and modulating OGA, facilitating its degradation and ubiquitination. Additionally, high-throughput compound library screening using three-dimensional protein structure analysis and drug screening identified a Food and Drug Administration drug, Y-39983 dihydrochloride, as a potent GEM sensitiser and UBR5 inhibitor. The combination of Y-39983 dihydrochloride and GEM attenuated tumour growth in a mouse xenograft tumour model. Collectively, these data demonstrated that UBR5 plays a pivotal role in the sensitisation of PC to GEM and provides a potential therapeutic strategy to overcome GEM resistance.

Dispersive effects and focused biodistribution of recombinant human hyaluronidase PH20: A locally acting and transiently active permeation enhancer.

BACKGROUND: Recombinant human hyaluronidase PH20 (rHuPH20) facilitates the dispersion and absorption of subcutaneously administered therapeutic agents. This study aimed to characterize the transient, local action of rHuPH20 in the subcutaneous (SC) space using focused biodistribution and dye dispersion studies conducted in mice. MATERIALS AND METHODS: To evaluate the biodistribution of rHuPH20, mice were intradermally administered rHuPH20 (80 U). The enzymatic activity of rHuPH20 was analyzed in the skin, lymph nodes, and plasma. Animal model sensitivity was determined by intravenous administration of rHuPH20 (80 U) to the tail vein. To evaluate local dispersion, mice received an intradermal injection of rHuPH20 followed by an intradermal injection of Trypan Blue dye at a contralateral site 45 minutes later. Dye dispersion was measured using a digital caliper. RESULTS: After intradermal rHuPH20 injection, enzymatic activity was detected within the skin near the injection site with levels decreasing rapidly after 15 minutes. There was no clear evidence of systemic exposure after administration of rHuPH20, and no discernible rHuPH20 activity was observed in lymph or plasma as a function of time after dosing. In the dye dispersion study, delivery of rHuPH20 at one site did not impact dye dispersion at a distal skin site. CONCLUSION: These observations support the classification of rHuPH20 as a transiently active and locally acting permeation enhancer.

Safety and pharmacokinetics of subcutaneous ceftriaxone administered with or without recombinant human hyaluronidase (rHuPH20) versus intravenous ceftriaxone administration in adult volunteers.

OBJECTIVE: To compare pharmacokinetics and safety of recombinant human hyaluronidase (rHuPH20)-facilitated subcutaneous (SC) ceftriaxone administration versus SC ceftriaxone preceded by SC saline placebo or intravenous (IV) ceftriaxone administration. RESEARCH DESIGN AND METHODS: This Phase I, two-part, placebo-controlled, crossover study was conducted in 54 healthy volunteers. In Part 1 (N = 24), subjects received 1 mL rHuPH20 (150 USP units) or placebo (0.9% sodium chloride) SC, followed by 1 or 2 g ceftriaxone (10-350 mg/mL). In Part 2 (N = 30), subjects received 1 g ceftriaxone at the Part 1 maximum tolerated concentration (MTC) administered either SC - preceded by SC rHuPH20 or placebo - or IV. Subjects were monitored for adverse events (AEs); blood samples were obtained (Part 2 only) during 48 hours post-dosing for ceftriaxone bioanalysis. MAIN OUTCOME MEASURES: Part 1 primary endpoint was the SC ceftriaxone (with or without rHuPH20) MTC. Pharmacokinetic parameters were determined in Part 2. Bioequivalence was based on maximum concentration (C(max)) and area under plasma concentration-time curve (AUC). RESULTS: The highest SC ceftriaxone concentration tested in Part 1 (350 mg/mL) was selected as the Part 2 MTC. In Part 2, median time to maximum concentration (t(max)) was 1 hour earlier (P < 0.0001), and C(max) was 12% higher (P < 0.0001) for ceftriaxone (350 mg/mL) administered via rHuPH20-facilitated SC versus SC preceded by placebo. IV ceftriaxone led to higher C(max) and shorter t(max) values than either SC treatment. Ceftriaxone exposure (AUC) was comparable among all three treatments. At least 1 AE was experienced by 100% of subjects after SC ceftriaxone and 76% after IV; most commonly reported AEs were infusion-site reactions. CONCLUSIONS: Ceftriaxone AUC did not differ significantly between the three administration routes. C(max) was higher and t(max) shorter with rHuPH20-facilitated SC than SC preceded by placebo. rHuPH20-facilitated SC ceftriaxone was generally well tolerated. This study is limited by evaluation of healthy adults and absence of repeated-dose groups.