Kinetic Referencing Allows Identification of Epigenetic Cytosine Modifications by Single-Molecule Hybridization Kinetics and Superresolution DNA-PAINT Microscopy.

We develop a DNA origami-based internal kinetic referencing system with a colocalized reference and target molecule to provide increased sensitivity and robustness for transient binding kinetics. To showcase this, we investigate the subtle changes in binding strength of DNA oligonucleotide hybrids induced by cytosine modifications. These cytosine modifications, especially 5-methylcytosine but also its oxidized derivatives, have been increasingly studied in the context of epigenetics. Recently revealed correlations of epigenetic modifications and disease also render them interesting biomarkers for early diagnosis. Internal kinetic referencing allows us to probe and compare the influence of the different epigenetic cytosine modifications on the strengths of 7-nucleotide long DNA hybrids with one or two modified nucleotides by single-molecule imaging of their transient binding, revealing subtle differences in binding times. Interestingly, the influence of epigenetic modifications depends on their position in the DNA strand, and in the case of two modifications, effects are additive. The sensitivity of the assay indicates its potential for the direct detection of epigenetic disease markers.

Healthy volunteers in first-in-human oncology drug development for small molecules.

This review provides tools to consider the inclusion of healthy volunteers (HVs) in first-in-human (FIH) oncology clinical trials with small molecules, including targeted and immunomodulatory agents, a strategy that was not envisioned with classic chemotherapy. To enable an FIH oncology trial in HVs compared to cancer patients (CPs), a robust nonclinical package must be generated, which includes toxicokinetic and pharmacokinetic studies, as well as more extensive safety pharmacology, toxicology and genotoxicity studies. This strategy could provide an early clinical characterization of the pharmacokinetic parameters and clinical safety profile in the absence of comorbidities and concomitant medication. It also avoids the ethical issue of administrating subtherapeutic doses to CPs, and could potentially help to accelerate the timelines of clinical drug development for patient care. That being said, stakeholders involved in these studies need to proceed with caution, fully understand the regulatory guidance and thoroughly evaluate the benefits and risks. This paper serves to address the regulatory guidance and other considerations needed when using healthy volunteers in early oncology trials.

Intragenomic Decarboxylation of 5-Carboxy-2'-deoxycytidine.

Cellular DNA is composed of four canonical nucleosides (dA, dC, dG and T), which form two Watson-Crick base pairs. In addition, 5-methylcytosine (mdC) may be present. The methylation of dC to mdC is known to regulate transcriptional activity. Next to these five nucleosides, the genome, particularly of stem cells, contains three additional dC derivatives, which are formed by stepwise oxidation of the methyl group of mdC with the help of Tet enzymes. These are 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC), and 5-carboxy-dC (cadC). It is believed that fdC and cadC are converted back into dC, which establishes an epigenetic control cycle that starts with methylation of dC to mdC, followed by oxidation and removal of fdC and cadC. While fdC was shown to undergo intragenomic deformylation to give dC directly, a similar decarboxylation of cadC was postulated but not yet observed on the genomic level. By using metabolic labelling, we show here that cadC decarboxylates in several cell types, which confirms that both fdC and cadC are nucleosides that are directly converted back to dC within the genome by C-C bond cleavage.

Nonclinical & clinical interface - extrapolation of nonclinical data to support Phase I clinical studies.

A review of the Investigator's Brochure and Clinical Study Reports for 58 non-oncology small molecule and biopharmaceutical drug candidates tested in a healthy volunteer subject population was conducted. Key findings were (1) a vital role for nonclinical pharmacology and toxicology testing was confirmed to allow setting of clinical starting dose and supporting use of highest dose based on No Observed Adverse Effect Levels (NOAELs), Pharmacologically Active Doses (PADs) and other approaches, (2) for clinical starting dose calculation, reference to the NOAEL was key, whether in calculation of a Maximum Recommended Starting Dose (MRSD), or by supporting PAD approaches (small molecules); or, through pharmacokinetic/pharmacodynamic (PK/PD) data modelling (biopharmaceuticals), (3) starting dose for small molecules was very conservative with human exposure >100- to 100-fold (46%) lower or between 10- and 100-fold (41%) lower than that seen at the NOAEL; high margins over exposure seen at NOAELs were also seen for biopharmaceuticals, (4) at the highest doses used, about 25% of studies for small molecules and 12% of studies for biopharmaceuticals showed exposure greater than that seen at the NOAEL and (5) adverse event evaluation showed that our current paradigm of moving from nonclinical testing into SAD/MAD Phase I testing is remarkably safe.

Progressive Myoclonic Epilepsies.

The treatment of progressive myoclonus epilepsy (PME) remains a major therapeutic challenge in neurology. Generalized convulsive seizures are often well controlled through classic antiepileptic drugs (AEDs) like valproate and clonazepam, whereas myoclonus, the main symptom that is affecting patients most in their daily life, is usually refractory to standard AEDs. Alternative therapy concepts have been and still are investigated. Among the new drugs, zonisamide and piracetam have shown the most promising results as add-on treatments. Other therapeutic approaches, like the use of antioxidants, 5-hydroxytryptophan (5-HTP), and baclofen should also be taken into consideration for the treatment of intractable cases of PME. Nonpharmacologic treatment options such as diet and physical therapy should always be considered, because they may save costs and side effects. In some instances, the occasional use of alcohol has shown beneficial effects.