Use of Methadone Versus Oxycodone to Facilitate Weaning of Parenteral Opioids in Critically Ill Adult Patients.

BACKGROUND: No previous literature has compared methadone with oxycodone for intravenous (IV) opioid weaning. OBJECTIVE: To determine if a weaning strategy using enteral methadone or oxycodone results in faster time to IV opioid discontinuation. METHODS: This was a single-center, retrospective, cohort medical record review of mechanically ventilated adults in an intensive care unit (ICU) who received a continuous IV infusion of fentanyl or hydromorphone for ≥72 hours and an enteral weaning strategy using either methadone or oxycodone from January 1, 2020, through December 31, 2021. Differences between groups were controlled for using Cox proportional hazards models. The primary outcome was time to continuous IV opioid discontinuation from the initiation of enteral opioids. Secondary outcomes included the primary endpoint stratified for COVID-19, duration of mechanical ventilation, ICU and hospital length of stay, and safety measures. RESULTS: Ninety-three patients were included, with 36 (38.7%) patients receiving methadone and 57 (61.3%) receiving oxycodone. Patients weaned using methadone received IV opioids significantly longer before the start of weaning (P = 0.04). However, those on methadone had a significantly faster time to discontinuation of IV opioids than those on oxycodone, mean (standard deviation) 104.7 (79.4) versus 158.3 hours (171.2), P = 0.04, and, at any time, were 1.89 times as likely to be weaned from IV opioids (hazard ratio, HR 1.89, 95% confidence interval, CI 1.16-3.07, P = 0.01). CONCLUSION AND RELEVANCE: This was the first study showing enteral methadone was associated with a shorter duration of IV opioids without differences in secondary outcomes compared with oxycodone. Prospective research is necessary to confirm this finding.

Encapsulating Cas9 into extracellular vesicles by protein myristoylation.

CRISPR/Cas9 genome editing is a very promising avenue for the treatment of a variety of genetic diseases. However, it is still very challenging to encapsulate CRISPR/Cas9 machinery for delivery. Protein N-myristoylation is an irreversible co/post-translational modification that results in the covalent attachment of the myristoyl-group to the N-terminus of a target protein. It serves as an anchor for a protein to associate with the cell membrane and determines its intracellular trafficking and activity. Extracellular vesicles (EVs) are secreted vesicles that mediate cell-cell communication. In this study, we demonstrate that myristoylated proteins were preferentially encapsulated into EVs. The octapeptide derived from the leading sequence of the N-terminus of Src kinase was a favourable substrate for N-myristoyltransferase 1, the enzyme that catalyzes myristoylation. The fusion of the octapeptide onto the N-terminus of Cas9 promoted the myristoylation and encapsulation of Cas9 into EVs. Encapsulation of Cas9 and sgRNA-eGFP inside EVs was confirmed using protease digestion assays. Additionally, to increase the transfection potential, VSV-G was introduced into the EVs. The encapsulated Cas9 in EVs accounted for 0.7% of total EV protein. Importantly, the EVs coated with VSV-G encapsulating Cas9/sgRNA-eGFP showed up to 42% eGFP knock out efficiency with limited off-target effects in recipient cells. Our study provides a novel approach to encapsulate CRISPR/Cas9 protein and sgRNA into EVs. This strategy may open an effective avenue to utilize EVs as vehicles to deliver CRISPR/Cas9 for genome-editing-based gene therapy.

Long-Chain Acyl-CoA Synthetase 4-Mediated Fatty Acid Metabolism Sustains Androgen Receptor Pathway-Independent Prostate Cancer.

Androgen deprivation therapy has led to elevated cases of androgen receptor (AR) pathway-independent prostate cancer with dysregulated fatty acid metabolism. However, it is unclear how prostate cancer cells sustain dysregulated fatty acid metabolism to drive AR-independent prostate cancer. Long-chain acyl-CoA synthetases (ACSL) catalyze the conversion of fatty acids into fatty acyl-CoAs that are required for fatty acid metabolism. In this study, we demonstrate that expression levels of ACSL3 and 4 were oppositely regulated by androgen-AR signaling in prostate cancer cells. AR served as a transcription suppressor to bind at the ACSL4 promoter region and inhibited its transcription. Inhibition of androgen-AR signaling significantly downregulated ACSL3 and PSA, but elevated ACSL4 levels. ACSL4 regulated a broad spectrum of fatty acyl-CoA levels, and its catalytic efficiency in fatty acyl-CoAs biosynthesis was about 1.9- to 4.3-fold higher than ACSL3. In addition, in contrast to ACSL3, ACSL4 significantly regulated global protein myristoylation or myristoylation of Src kinase in prostate cancer cells. Knockdown of ACSL4 inhibited the proliferation, migration, invasion, and xenograft growth of AR-independent prostate cancer cells. Our results suggest that the surge of ACSL4 levels by targeting AR signaling increases fatty acyl-CoAs biosynthesis and protein myristoylation, indicating the opposite, yet complementary or Yin-Yang regulation of ACSL3 and 4 levels in sustaining fatty acid metabolism when targeting androgen-AR signaling. This study reveals a mechanistic understanding of ACSL4 as a potential therapeutic target for treatment of AR-independent prostate cancer. IMPLICATIONS: AR coordinately regulates the expression of ACSL3 and ACSL4, such that AR pathway-independent prostate tumors become dependent on ACSL4-mediated fatty acid metabolism.

Blocking Myristoylation of Src Inhibits Its Kinase Activity and Suppresses Prostate Cancer Progression.

Protein N-myristoylation enables localization to membranes and helps maintain protein conformation and function. N-myristoyltransferases (NMT) catalyze co- or posttranslational myristoylation of Src family kinases and other oncogenic proteins, thereby regulating their function. In this study, we provide genetic and pharmacologic evidence that inhibiting the N-myristoyltransferase NMT1 suppresses cell-cycle progression, proliferation, and malignant growth of prostate cancer cells. Loss of myristoylation abolished the tumorigenic potential of Src and its synergy with androgen receptor in mediating tumor invasion. We identified the myristoyl-CoA analogue B13 as a small-molecule inhibitor of NMT1 enzymatic activity. B13 exposure blocked Src myristoylation and Src localization to the cytoplasmic membrane, attenuating Src-mediated oncogenic signaling. B13 exerted its anti-invasive and antitumor effects against prostate cancer cells, with minimal toxic side-effects in vivo Structural optimization based on structure-activity relationships enabled the chemical synthesis of LCL204, with enhanced inhibitory potency against NMT1. Collectively, our results offer a preclinical proof of concept for the use of protein myristoylation inhibitors as a strategy to block prostate cancer progression. Cancer Res; 77(24); 6950-62. ©2017 AACR.