Stability of Opened Durvalumab (IMFINZI) Vials. The Beginning of the End of Costly Product Wastage?

Durvalumab is a monoclonal antibody approved for the treatment of lung, urothelial and biliary tract cancers. Durvalumab is supplied in vials as a solution containing no preservatives. Monographs recommend single use of durvalumab vials, and that any leftovers be discarded within 24 h. Thus, significant portions of unused product from opened vials are wasted on a daily basis, generating considerable financial losses. The objective of the present study was to assess the physicochemical and microbiological stability of durvalumab vials kept at 4 °C or room temperature, at 7 and 14 days after opening. Following pH and osmolality measurements, turbidity and submicronic aggregation of durvalumab solution were evaluated by spectrophotometry and dynamic light scattering, respectively. Moreover, steric exclusion high performance liquid chromatography (SE-HPLC), ion exchange HPLC (IEX-HPLC) and peptide mapping HPLC were used to respectively assess aggregation/fragmentation, charge distribution and primary structure of durvalumab. Microbiological stability of durvalumab was evaluated by incubation of vial leftovers on blood agar. All experiments showed physicochemical and microbiological stability of durvalumab vial leftovers for at least 14 days when aseptically handled and kept at either 4 °C or at room temperature. These results suggest the possible extension of utilization of durvalumab vial leftovers well beyond 24 h.

Dual-Labelled Nanoparticles Inform on the Stability of Fluorescent Labels In Vivo.

Fluorescent labelling is commonly used to monitor the biodistribution of nanomedicines. However, meaningful interpretation of the results requires that the fluorescent label remains attached to the nanomedicine. In this work, we explore the stability of three fluorophores (BODIPY650, Cyanine 5 and AZ647) attached to polymeric hydrophobic biodegradable anchors. Using dual-labelled poly(ethylene glycol)-b-poly(lactic acid) (PEG-PLA) nanoparticles that are both radioactive and fluorescent, we investigated how the properties of the fluorophores impact the stability of the labelling in vitro and in vivo. Results suggest that the more hydrophilic dye (AZ647) is released faster from nanoparticles, and that this instability results in misinterpretation of in vivo data. While hydrophobic dyes are likely more suitable to track nanoparticles in biological environments, quenching of the fluorescence inside the nanoparticles can also introduce artefacts. Altogether, this work raises awareness about the importance of stable labelling methods when investigating the biological fate of nanomedicines.

The mechanisms of anti-PEG immune response are different in the spleen and the lymph nodes.

Polyethylene glycol (PEG) is a common ingredient in nanomedicines and pharmaceuticals. Recent studies show that approximately 20-70% of humans have anti-PEG antibodies that can recognize the polymer. Because these anti-PEG antibodies can reduce the effectiveness of certain PEGylated therapeutics, understanding how these immunoglobulins are produced is important. In this work, we investigate the mechanisms of the anti-PEG immune response, following the injection of polymeric nanoparticles by different routes of administration. We observed that the extent of systemic absorption and splenic deposition cannot predict the production of anti-PEG IgM - possibly because redundant biological pathways can be involved. Data obtained by surgically removing the spleen or depleting the complement activity suggest that the mechanisms behind the anti-PEG immune response differ between intravenous and subcutaneous injections. While B cells from the spleen appear to necessitate complement proteins to interact with nanoparticles, internalization by follicular B cells from the lymph nodes is unaffected by depletion of the cascade. This study confirms that the biological mechanisms involved in the immune recognition of nanomedicines varies based on the administration route. This knowledge can be utilized to use nanomedicines to engage the immune system in differentiated ways.

Size Exclusion of Radioactive Polymers (SERP) informs on the biodegradation of trimethyl chitosan and biodegradable polymer nanoparticles in vitro and in vivo.

Preparation of drug delivery systems and nanomedicines necessitates the use of biocompatible excipients that are readily eliminated from the body. The systematic preclinical development of novel materials requires tools to evaluate their pharmacokinetics, biodistribution and excretion. Herein, we propose a technique called Size Exclusion of Radioactive Polymers (SERP) to trail the disposition of a radiolabeled polymer and its nanoparticles using chromatography in the presence of complex biological media such as blood, urine and feces. Trimethyl chitosan (TMC) is a polysaccharide of natural origin showing promise for controlled and targeted drug delivery applications. SERP was used to monitor degradation of radiolabeled TMC and its nanoparticles in vitro in the presence of strong acid, enzymes released by macrophages, as well as in vivo after administration to rats. Excretion of the radiolabeled TMC nanoparticles in urine and feces was monitored for 14 days after dosing to healthy rats, confirming that the polymer could be readily eliminated from the body. This work demonstrates the ability of SERP to understand the biological journey of biomaterials in vivo. Paving the way to understand the fate of polymers and nanoparticles in complex environments, the technique might facilitate the development of safer and better tolerated nanomedicines.

Pituitary cell translation and secretory capacities are enhanced cell autonomously by the transcription factor Creb3l2.

Translation is a basic cellular process and its capacity is adapted to cell function. In particular, secretory cells achieve high protein synthesis levels without triggering the protein stress response. It is unknown how and when translation capacity is increased during differentiation. Here, we show that the transcription factor Creb3l2 is a scaling factor for translation capacity in pituitary secretory cells and that it directly binds ~75% of regulatory and effector genes for translation. In parallel with this cell-autonomous mechanism, implementation of the physiological UPR pathway prevents triggering the protein stress response. Knockout mice for Tpit, a pituitary differentiation factor, show that Creb3l2 expression and its downstream regulatory network are dependent on Tpit. Further, Creb3l2 acts by direct targeting of translation effector genes in parallel with signaling pathways that otherwise regulate protein synthesis. Expression of Creb3l2 may be a useful means to enhance production of therapeutic proteins.

Interplay between ShcA Signaling and PGC-1α Triggers Targetable Metabolic Vulnerabilities in Breast Cancer.

The ShcA adaptor protein transduces oncogenic signals downstream of receptor tyrosine kinases. We show here that breast tumors engage the ShcA pathway to increase their metabolism. ShcA signaling enhanced glucose catabolism through glycolysis and oxidative phosphorylation, rendering breast cancer cells critically dependent on glucose. ShcA signaling simultaneously increased the metabolic rate and flexibility of breast cancer cells by inducing the PGC-1α transcriptional coactivator, a central regulator of mitochondrial metabolism. Breast tumors that engaged ShcA signaling were critically dependent on PGC-1α to support their increased metabolic rate. PGC-1α deletion drastically delayed breast tumor onset in an orthotopic mouse model, highlighting a key role for PGC-1α in tumor initiation. Conversely, reduced ShcA signaling impaired both the metabolic rate and flexibility of breast cancer cells, rendering them reliant on mitochondrial oxidative phosphorylation. This metabolic reprogramming exposed a targetable metabolic vulnerability, leading to a sensitization of breast tumors to inhibitors of mitochondrial complex I (biguanides). Genetic inhibition of ShcA signaling in the Polyoma virus middle T (MT) breast cancer mouse model sensitized mammary tumors to biguanides during the earliest stages of breast cancer progression. Tumor initiation and growth were selectively and severely impaired in MT/ShcA-deficient animals. These data demonstrate that metabolic reprogramming is a key component of ShcA signaling and serves an unappreciated yet vital role during breast cancer initiation and progression. These data further unravel a novel interplay between ShcA and PGC-1α in the coordination of metabolic reprogramming and demonstrate the sensitivity of breast tumors to drugs targeting oxidative phosphorylation.Significance: This study uncovers a previously unrecognized mechanism that links aberrant RTK signaling with metabolic perturbations in breast cancer and exposes metabolic vulnerabilities that can be targeted by inhibitors of oxidative phosphorylation. Cancer Res; 78(17); 4826-38. ©2018 AACR.

PGC-1α Promotes Breast Cancer Metastasis and Confers Bioenergetic Flexibility against Metabolic Drugs.

Metabolic adaptations play a key role in fueling tumor growth. However, less is known regarding the metabolic changes that promote cancer progression to metastatic disease. Herein, we reveal that breast cancer cells that preferentially metastasize to the lung or bone display relatively high expression of PGC-1α compared with those that metastasize to the liver. PGC-1α promotes breast cancer cell migration and invasion in vitro and augments lung metastasis in vivo. Pro-metastatic capabilities of PGC-1α are linked to enhanced global bioenergetic capacity, facilitating the ability to cope with bioenergetic disruptors like biguanides. Indeed, biguanides fail to mitigate the PGC-1α-dependent lung metastatic phenotype and PGC-1α confers resistance to stepwise increases in metformin concentration. Overall, our results reveal that PGC-1α stimulates bioenergetic potential, which promotes breast cancer metastasis and facilitates adaptation to metabolic drugs.

PRL2 links magnesium flux and sex-dependent circadian metabolic rhythms.

Magnesium (Mg2+) plays pleiotropic roles in cellular biology, and it is essentially required for all living organisms. Although previous studies demonstrated intracellular Mg2+ levels were regulated by the complex of phosphatase of regenerating liver 2 (PRL2) and Mg2+ transporter of cyclin M (CNNMs), physiological functions of PRL2 in whole animals remain unclear. Interestingly, Mg2+ was recently identified as a regulator of circadian rhythm-dependent metabolism; however, no mechanism was found to explain the clock-dependent Mg2+ oscillation. Herein, we report PRL2 as a missing link between sex and metabolism, as well as clock genes and daily cycles of Mg2+ fluxes. Our results unveil that PRL2-null animals displayed sex-dependent alterations in body composition, and expression of PRLs and CNNMs were sex- and circadian time-dependently regulated in brown adipose tissues. Consistently, PRL2-KO mice showed sex-dependent alterations in thermogenesis and in circadian energy metabolism. These physiological changes were associated with an increased rate of uncoupled respiration with lower intracellular Mg2+ in PRL2-KO cells. Moreover, PRL2 deficiency causes inhibition of the ATP citrate lyase axis, which is involved in fatty acid synthesis. Overall, our findings support that sex- and circadian-dependent PRL2 expression alter intracellular Mg2+ levels, which accordingly controls energy metabolism status.

The complete targeted profile of the organic acid intermediates of the citric acid cycle using a single stable isotope dilution analysis, sodium borodeuteride reduction and selected ion monitoring GC/MS.

The quantitative profiling of the organic acid intermediates of the citric acid cycle (CAC) presents a challenge due to the lack of commercially available internal standards for all of the organic acid intermediates. We developed an analytical method that enables the quantitation of all the organic acids in the CAC in a single stable isotope dilution GC/MS analysis with deuterium-labeled analogs used as internal standards. The unstable α-keto acids are rapidly reduced with sodium borodeuteride to the corresponding stable α-deutero-α-hydroxy acids and these, along with their unlabeled analogs and other CAC organic acid intermediates, are converted to their tert-butyldimethylsilyl derivatives. Selected ion monitoring is employed with electron ionization. We validated this method by treating an untransformed mouse mammary epithelial cell line with well-known mitochondrial toxins affecting the electron transport chain and ATP synthase, which resulted in profound perturbations of the concentration of CAC intermediates.

mTORC1 controls mitochondrial activity and biogenesis through 4E-BP-dependent translational regulation.

mRNA translation is thought to be the most energy-consuming process in the cell. Translation and energy metabolism are dysregulated in a variety of diseases including cancer, diabetes, and heart disease. However, the mechanisms that coordinate translation and energy metabolism in mammals remain largely unknown. The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) stimulates mRNA translation and other anabolic processes. We demonstrate that mTORC1 controls mitochondrial activity and biogenesis by selectively promoting translation of nucleus-encoded mitochondria-related mRNAs via inhibition of the eukaryotic translation initiation factor 4E (eIF4E)-binding proteins (4E-BPs). Stimulating the translation of nucleus-encoded mitochondria-related mRNAs engenders an increase in ATP production capacity, a required energy source for translation. These findings establish a feed-forward loop that links mRNA translation to oxidative phosphorylation, thereby providing a key mechanism linking aberrant mTOR signaling to conditions of abnormal cellular energy metabolism such as neoplasia and insulin resistance.

PGC-1α promotes the growth of ErbB2/Neu-induced mammary tumors by regulating nutrient supply.

Cancer cells display an increased reliance on glycolysis despite the presence of sufficient oxygen levels to support mitochondrial functions. In this study, we asked whether ameliorating mitochondrial functions in cancer cells might limit their proliferative capacity. Specifically, we increased mitochondrial metabolism in a murine cellular model of ErbB2/Neu-induced breast cancer by ectopically expressing the transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a master regulator of mitochondrial metabolism. As predicted, ErbB2/Neu cells ectopically expressing PGC-1α displayed an increased level of mitochondrial metabolism and reduced proliferative capacity in vitro, compared with controls. In contrast, ErbB2/Neu cells ectopically expressing PGC-1α formed larger tumors in vivo. These tumors exhibited increased concentrations of glucose and the angiogenic factor VEGF as well as higher expression of ErbB2/Neu compared with controls. We discovered that ErbB2/Neu levels were sensitive to nutrient availability, such that reduced glucose concentrations resulted in diminished ErbB2/Neu protein levels. Therefore, our data indicate that PGC-1α prevents the nutrient-mediated downregulation of ErbB2/Neu in tumors by increasing glucose supply. Mechanistic investigations revealed that the regulation of ErbB2/Neu levels by glucose was mediated by the unfolded protein response (UPR). Incubation of ErbB2/Neu-induced breast cancer cells in limited glucose concentrations or with drugs that activate the UPR led to significant reductions in ErbB2/Neu protein levels. Also, ErbB2/Neu-induced tumors ectopically expressing PGC-1α displayed lowered UPR activation compared with controls. Together, our findings uncover an unexpected link between PGC-1α-mediated nutrient availability, UPR, and ErbB2/Neu levels.

Expression analysis and essential role of the putative tyrosine phosphatase His-domain-containing protein tyrosine phosphatase (HD-PTP).

The putative tyrosine phosphatase HD-PTP, encoded by the protein-tyrosine-phosphatase-n23 (Ptpn23) gene, has been described as a tumor suppressor candidate gene. However, its physiological roles and detailed expression profiles are poorly defined. To investigate HD-PTP functions, we generated a mouse model in which the Ptpn23 locus was disrupted by an in-frame insertion of a beta-galactosidase-neomycin-phosphotransferase II (beta-geo) cassette. This insertion leads to the expression of a catalytically inactive truncated protein preserving only the uncharacterized N-terminal BRO1-like domain in fusion with beta-geo under the control of the endogenous promoter. Here we report that homozygous gene deletion is lethal around embryonic day 9.5, suggesting that Ptpn23 is an essential requirement for early stages of embryonic development. Taking advantage of the beta-galactosidase insertion into the Ptpn23 locus, we define the precise Ptpn23 expression pattern by performing X-gal staining at different stages of mouse development. Our results show that Ptpn23 is expressed early during mouse development and that its expression is maintained in adult tissues, markedly in the epithelial cells of many organs.

The proinflammatory actions of angiotensin II are dependent on p65 phosphorylation by the IkappaB kinase complex.

The vasoactive hormone angiotensin II (Ang II) probably triggers inflammatory cardiovascular diseases by activating transcription factors such as NF-kappaB. We describe here a novel mode of NF-kappaB activation in cultured vascular smooth muscle cells exposed to Ang II. Ang II treatment resulted in an increase in the phosphotransferase activity of the IKK complex, which was mediated through the AT1 receptor subtype. The typical phosphorylation and proteasome-dependent degradation of the NF-kappaB inhibitor IkappaBalpha were not observed. Rather, Ang II treatment of vascular smooth muscle cells led to the phosphorylation of p65 on serine 536, a signal detected in both the cytoplasm and the nuclear compartments. The use of pharmacological inhibitors that inhibit the activation of MEK by Ang II revealed that phosphorylation of p65 on serine 536 did not require the MEK-ERK-RSK signaling pathway. On the other hand, specifically targeting the IKKbeta subunit of the IKK complex by overexpression of a dominant negative version of IKKbeta (IKKbeta K44A) or silencing RNA technology demonstrated that the IKKbeta subunit of the IKK complex was responsible for the detected phosphoserine 536 signal in Ang II-treated cells. Characterization of the signaling pathway leading to activation of the IKK complex by Ang II revealed that neither epidermal growth factor receptor transactivation nor the phosphatidylinositol 3-kinase-AKT signaling cascade were involved. Collectively, our data demonstrate that the proinflammatory activity of Ang II is independent of the classical pathway leading to IkappaBalpha phosphorylation and degradation but clearly depends on the recruitment of an IKK complex signaling cascade leading to phosphorylation of p65 on serine 536.