Strategies for the production of long-acting therapeutics and efficient drug delivery for cancer treatment

Protein therapeutics play a significant role in treating many diseases. They, however, suffer from patient's proteases degradation and antibody neutralization which lead to short plasma half-lives. One of the ways to overcome these pitfalls is the frequent injection of the drug albeit at the cost of patient compliance which affects the quality of life of patients. There are several techniques available to extend the half-life of therapeutics. Two of the most common protocols are PEGylation and fusion with human serum albumin. These two techniques improve stability, reduce immunogenicity, and increase drug resistance to proteases. These factors lead to the reduction of injection frequency which increases patient compliance and improve quality of life. Both techniques have already been used in many FDA approved drugs. This review describes many technologies to produce long-acting drugs with the attention of PEGylation and the genetic fusion with human serum albumin. The report also discusses the latest modified therapeutics in the field and their application in cancer therapy. We compare the modification methods and discuss the pitfalls of these modified drugs

Production of "biobetter" variants of glucarpidase with enhanced enzyme activity

Glucarpidase, also known as carboxypeptidase G(2), is a Food and Drug Administration-approved enzyme used in targeted cancer strategies such as antibody-directed enzyme prodrug therapy (ADEPT). It is also used in drug detoxification when cancer patients have excessive levels of the anti-cancer agent methotrexate. The application of glucarpidase is limited by its potential immunogenicity and limited catalytic efficiency. To overcome these pitfalls, mutagenesis was applied to the glucarpidase gene of Pseudomonas sp. strain RS-16 to isolate three novels "biobetter" variants with higher specific enzyme activity. DNA sequence analysis of the genes for the variants showed that each had a single point mutation, resulting in the amino acid substitutions: I100 T, G123S and T239 A. K-m, V-max and K-cat measurements confirmed that each variant had increased catalytic efficiency relative to wild type glucarpidase. Additionally, circular dichroism studies indicated that they had a higher alpha-helical content relative to the wild type enzyme. However, three different software packages predicted that they had reduced protein stability, which is consistent with having higher activities as a tradeoff. The novel glucarpidase variants presented in this work could pave the way for more efficient drug detoxification and might allow dose escalation during chemotherapy. They also have the potential to increase the efficiency of ADEPT and to reduce the number of treatment cycles, thereby reducing the risk that patients will develop antibodies to glucarpidase

Genome-wide DNA methylation analysis for diabetic nephropathy in type 1 diabetes mellitus

BACKGROUND: Diabetic nephropathy is a serious complication of diabetes mellitus and is associated with considerable morbidity and high mortality. There is increasing evidence to suggest that dysregulation of the epigenome is involved in diabetic nephropathy. We assessed whether epigenetic modification of DNA methylation is associated with diabetic nephropathy in a case-control study of 192 Irish patients with type 1 diabetes mellitus (T1D). Cases had T1D and nephropathy whereas controls had T1D but no evidence of renal disease. METHODS: We performed DNA methylation profiling in bisulphite converted DNA from cases and controls using the recently developed Illumina Infinium(R) HumanMethylation27 BeadChip, that enables the direct investigation of 27,578 individual cytosines at CpG loci throughout the genome, which are focused on the promoter regions of 14,495 genes. RESULTS: Singular Value Decomposition (SVD) analysis indicated that significant components of DNA methylation variation correlated with patient age, time to onset of diabetic nephropathy, and sex. Adjusting for confounding factors using multivariate Cox-regression analyses, and with a false discovery rate (FDR) of 0.05, we observed 19 CpG sites that demonstrated correlations with time to development of diabetic nephropathy. Of note, this included one CpG site located 18 bp upstream of the transcription start site of UNC13B, a gene in which the first intronic SNP rs13293564 has recently been reported to be associated with diabetic nephropathy. CONCLUSION: This high throughput platform was able to successfully interrogate the methylation state of individual cytosines and identified 19 prospective CpG sites associated with risk of diabetic nephropathy. These differences in DNA methylation are worthy of further follow-up in replication studies using larger cohorts of diabetic patients with and without nephropathy

Hard Two-Photon Contribution to Elastic Lepton-Proton Scattering: Determined by the OLYMPUS Experiment

The OLYMPUS collaboration reports on a precision measurement of the positron-proton to electron-proton elastic cross section ratio, $R_{2\gamma}$, a direct measure of the contribution of hard two-photon exchange to the elastic cross section. In the OLYMPUS measurement, 2.01~GeV electron and positron beams were directed through a hydrogen gas target internal to the DORIS storage ring at DESY. A toroidal magnetic spectrometer instrumented with drift chambers and time-of-flight scintillators detected elastically scattered leptons in coincidence with recoiling protons over a scattering angle range of $\approx 20\degree$ to $80\degree$. The relative luminosity between the two beam species was monitored using tracking telescopes of interleaved GEM and MWPC detectors at $12\degree$, as well as symmetric M{\o}ller/Bhabha calorimeters at $1.29\degree$. A total integrated luminosity of 4.5~fb$^{-1}$ was collected. In the extraction of $R_{2\gamma}$, radiative effects were taken into account using a Monte Carlo generator to simulate the convolutions of internal bremsstrahlung with experiment-specific conditions such as detector acceptance and reconstruction efficiency. The resulting values of $R_{2\gamma}$, presented here for a wide range of virtual photon polarization $0.456<\epsilon<0.978$, are smaller than some hadronic two-photon exchange calculations predict, but are in reasonable agreement with a subtracted dispersion model and a phenomenological fit to the form factor data.Comment: 5 pages, 3 figures, 2 table

Insulin-like growth factor 1 signaling is essential for mitochondrial biogenesis and mitophagy in cancer cells

Mitochondrial activity and metabolic reprogramming influence the phenotype of cancer cells and resistance to targeted therapy. We previously established that an insulin-like growth factor 1 (IGF-1)-inducible mitochondrial UTP carrier (PNC1/SLC25A33) promotes cell growth. This prompted us to investigate whether IGF signaling is essential for mitochondrial maintenance in cancer cells and whether this contributes to therapy resistance. Here we show that IGF-1 stimulates mitochondrial biogenesis in a range of cell lines. In MCF-7 and ZR75.1 breast cancer cells, IGF-1 induces peroxisome proliferator–activated receptor γ coactivator 1β (PGC-1β) and PGC-1α–related coactivator (PRC). Suppression of PGC-1β and PRC with siRNA reverses the effects of IGF-1 and disrupts mitochondrial morphology and membrane potential. IGF-1 also induced expression of the redox regulator nuclear factor-erythroid-derived 2-like 2 (NFE2L2 alias NRF-2). Of note, MCF-7 cells with acquired resistance to an IGF-1 receptor (IGF-1R) tyrosine kinase inhibitor exhibited reduced expression of PGC-1β, PRC, and mitochondrial biogenesis. Interestingly, these cells exhibited mitochondrial dysfunction, indicated by reactive oxygen species expression, reduced expression of the mitophagy mediators BNIP3 and BNIP3L, and impaired mitophagy. In agreement with this, IGF-1 robustly induced BNIP3 accumulation in mitochondria. Other active receptor tyrosine kinases could not compensate for reduced IGF-1R activity in mitochondrial protection, and MCF-7 cells with suppressed IGF-1R activity became highly dependent on glycolysis for survival. We conclude that IGF-1 signaling is essential for sustaining cancer cell viability by stimulating both mitochondrial biogenesis and turnover through BNIP3 induction. This core mitochondrial protective signal is likely to strongly influence responses to therapy and the phenotypic evolution of cancer

Dairy consumption and cardiometabolic diseases: systematic review and updated meta-analyses of prospective cohort studies

Purpose of Review Dairy products contain both beneficial and harmful nutrients in relation to cardiometabolic diseases. Here, we provide the latest scientific evidence regarding the relationship between dairy products and cardiometabolic diseases by reviewing the literature and updating meta-analyses of observational studies. Recent Findings We updated our previous meta-analyses of cohort studies on type 2 diabetes, coronary heart disease (CHD), and stroke with nine studies and confirmed previous results. Total dairy and low-fat dairy (per 200 g/d) were inversely associated with a 3–4% lower risk of diabetes. Yogurt was non-linearly inversely associatedwith diabetes (RR = 0.86, 95%CI: 0.83–0.90 at 80 g/ d). Total dairy and milk were not associated with CHD (RR~1.0). An increment of 200 g of daily milk intake was associated with an 8% lower risk of stroke. Summary The latest scientific evidence confirmed neutral or beneficial associations between dairy products and risk of cardiometabolic diseases

A Phase I, First-in-Human Study of GSK2849330, an Anti-HER3 Monoclonal Antibody, in HER3-Expressing Solid Tumors

Background GSK2849330, an anti-HER3 monoclonal antibody that blocks HER3/Neuregulin 1 (NRG1) signaling in cancer cells, is engineered for enhanced antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. This phase I, first-in-human, open-label study assessed the safety, pharmacokinetics (PK), pharmacodynamics, and preliminary activity of GSK2849330 in patients with HER3-expressing advanced solid tumors. Patients and Methods Patients with various tumor types were prospectively selected for HER3 expression by immunohistochemistry; a subset was also screened for NRG1 mRNA expression. In the dose-escalation phase, patients received GSK2849330 1.4-30 mg/kg every 2 weeks, or 3 mg/kg or 30 mg/kg weekly, intravenously (IV). In the dose-expansion phase, patients received 30 mg/kg GSK2849330 IV weekly. Results Twenty-nine patients with HER3-expressing cancers, of whom two expressed NRG1, received GSK2849330 (dose escalation: n = 18, dose expansion: n = 11). GSK2849330 was well tolerated. No dose-limiting toxicities were observed. The highest dose, of 30 mg/kg weekly, expected to provide full target engagement, was selected for dose expansion. Treatment-emergent adverse events (AEs) were mostly grade 1 or 2. The most common AEs were diarrhea (66%), fatigue (62%), and decreased appetite (31%). Dose-proportional plasma exposures were achieved, with evidence of HER3 inhibition in paired tissue biopsies. Of 29 patients, only 1 confirmed partial response, lasting 19 months, was noted in a patient with CD74-NRG1-rearranged non-small cell lung cancer (NSCLC). Conclusion GSK2849330 demonstrated a favorable safety profile, dose-proportional PK, and evidence of target engagement, but limited antitumor activity in HER3-expressing cancers. The exceptional response seen in a patient with CD74-NRG1-rearranged NSCLC suggests further exploration in NRG1-fusion-positive cancers. Implications for Practice This first-in-human study confirms that GSK2849330 is well tolerated. Importantly, across a variety of HER3-expressing advanced tumors, prospective selection by HER3/NRG1 expression alone was insufficient to identify patients who could benefit from treatment with this antibody-dependent cell-mediated cytotoxicity- and complement-dependent cytotoxicity-enhanced anti-HER3 antibody. The only confirmed durable response achieved was in a patient with CD74-NRG1-rearranged lung cancer. This highlights the potential utility of screening for NRG1 fusions prospectively across tumor types to enrich potential responders to anti-HER3 agents in ongoing trials

Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization

The effects of photocaged nucleosides on the DNA polymerization reaction was investigated, finding that most polymerases are unable to recognize and read through the presence of a single caging group on the DNA template. Based on this discovery, a new method of introducing mutations into plasmid DNA via a light-mediated mutagenesis protocol was developed. This methodology is advantageous over several common approaches in that it requires the use of only two polymerase chain reaction primers, and does not require any restriction sites or use of restriction enzymes. Additionally, this approach enables not only site-directed mutations, but also the insertion of DNA strands of any length into plasmids and the deletion of entire genes from plasmids