New Technologies to Prolong Life-time of Peptide and Protein Drugs In vivo

Most peptide and protein drugs are short-lived species in vivo with a circulatory half-life of several minutes. This is particularly valid for non-glycosylated proteins with a molecular mass of less than 50 kDa. Since peptide/protein drugs are not absorbed orally, prolonged maintenance of therapeutically active drugs in the circulatory system is of primary clinical importance. Another major obstacle of injected polypeptide drugs is the elevated concentration of 100–1000 times above the therapeutical level that may be present in the circulatory system shortly after administration. Such overdosing may lead to undesirable side effects such as over-stimulation or down-regulation of receptor sites. In this review we describe two new strategies that overcome these two problems of systemically injected peptide/protein drugs. The first strategy includes Fmoc and FMS derivatization of peptides, proteins and low molecular-weight drugs, converting them to inactive prodrugs that undergo reactivation with desirable pharmacokinetic patterns in body fluids. Based on this Fmoc/FMS-technology, we have developed a second strategy, reversible pegylation. Inactive pegylated peptide/protein drugs release the native active parental molecule at slow rates, and in homogeneous fashion under physiological conditions, thus facilitating prolonged therapeutic effects, following a single administration.     

hERG K+ channel blockade by the antipsychotic drug thioridazine: An obligatory role for the S6 helix residue F656

The phenothiazine antipsychotic agent thioridazine has been linked with prolongation of the QT interval on the electrocardiogram, ventricular arrhythmias, and sudden death. Although thioridazine is known to inhibit cardiac hERG K(+) channels there is little mechanistic information on this action. We have investigated in detail hERG K(+) channel current (I(hERG)) blockade by thioridazine and identified a key molecular determinant of blockade. Whole-cell I(hERG) measurements were made at 37 degrees C from human embryonic kidney (HEK-293) cells expressing wild-type and mutant hERG channels. Thioridazine inhibited I(hERG) tails at -40mV following a 2s depolarization to +20mV with an IC(50) value of 80nM. Comparable levels of I(hERG) inhibition were seen with physiological command waveforms (ventricular and Purkinje fibre action potentials). Thioridazine block of I(hERG) was only weakly voltage-dependent, though the time dependence of I(hERG) inhibition indicated contingency of blockade upon channel gating. The S6 helix point mutation F656A almost completely abolished, and the Y652A mutation partially attenuated, I(hERG) inhibition by thioridazine. In summary, thioridazine is one of the most potent hERG K(+) channel blockers amongst antipsychotics, exhibiting characteristics of a preferential open/activated channel blocker and binding at a high affinity site in the hERG channel pore.     

High affinity HERG K(+) channel blockade by the antiarrhythmic agent dronedarone: resistance to mutations of the S6 residues Y652 and F656

Pharmacological inhibition of human-ether-a-go-go-related gene (HERG) K(+) channels by structurally and therapeutically diverse drugs is associated with the 'acquired' form of long QT syndrome and with potentially lethal cardiac arrhythmias. Two aromatic amino-acid residues (Y652 and F656) on the inner (S6) helices are considered to be key constituents of a high affinity drug binding site within the HERG channel pore cavity. Using wild-type (WT) and mutant HERG channels expressed in mammalian cell lines, we have investigated HERG channel current (I(HERG)) blockade at 37+/-1 degrees C by dronedarone (DRONED), a non-iodinated analogue of the Class III antiarrhythmic agent amiodarone (AMIOD). Under our conditions WT I(HERG) tails, measured at -40 mV following activating pulses to +30 mV, were blocked with IC(50) values of approximately 59 and 70 nM for DRONED and AMIOD, respectively. I(HERG) inhibition by DRONED was contingent upon channel gating, with block developing rapidly on membrane depolarization, but with no preference for activated over inactivated channels. High external [K(+)] (94 mM) reduced the potency of I(HERG) inhibition by both DRONED and AMIOD. Strikingly, mutagenesis to alanine of the S6 residue F656 (F656A) failed to eliminate blockade by both DRONED and AMIOD, whilst Y652A had comparatively little effect on DRONED but some effect on AMIOD. These findings demonstrate that high affinity drug blockade of I(HERG) can occur without a strong dependence on the Y652 and F656 aromatic amino-acid residues.     

Assessment of QT liabilities in drug development

Since the publication, in 1997, of the CPMP (Committee for Proprietary Medicinal Products) Points to Consider document on "The assessment of potential for QT prolongation by non-cardiovascular medicinal products," both regulatory bodies and the pharmaceutical industry have paid increasing attention to the conduct of careful preclinical studies on the subject. Regulatory attention has focused on the drafting of Safety Pharmacology guidelines through the ICH (International Conference on Harmonization) process, which resulted in approval by the ICH and acceptance by the three main regions (USA, Europe, and Japan) of the ICH S7A guideline. The guideline does not deal only with cardiovascular studies and does not provide guidance on QT investigations. This part has been deferred to a second guideline (ICH S7B). Nevertheless, pharmaceutical companies have implemented screening strategies aimed at selecting compounds that do not present QT liabilities. These strategies can differ according to the pharmaceutical class, while experimental models differ according to the stage of development of the compound. Several in vitro models are employed in discovery (radioligand binding, high-throughput patch clamp, efflux, and fluorescence assays). These models, coupled with in silico methods, allow companies to screen a high number of compounds. Other in vitro models, applied later in the R&D process (action potential duration, APD, in Purkinje fibers or papillary muscle and the isolated heart) are useful in better describing the activity of compounds on cardiac ion channels. The most robust and accepted in vivo test is represented by telemetry studies in conscious non-rodents.     

From dimictic to monomictic: Empirical evidence of thermal regime transitions in three deep alpine lakes in Austria induced by climate change

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Prolongation of the QT interval and post-extrasystolic augmentation of the TU-wave during emotional stress

We present a case of a 25-year-old woman with multiple blackouts and no structural heart disease, with abnormal T-U waves and borderline QT interval on her resting electrocardiogram. During emotional stress she developed frequent monomorphic ventricular premature beats, with characteristic changes of the sinus complexes immediately following the premature beats, namely augmentation and greater degree of merging of the T and U waves and QTc interval prolongation. The changes alert about the possibility of congenital long QT syndrome, specifically genotype 2 or 1.     

Sex differences in murine cardiac pathophysiology with hyperoxia exposure

Hyperoxia (>90% oxygen) is commonly implemented in mechanically ventilated patients. Reports suggest that hyperoxia is directly associated with in-hospital mortality in ventilated patients. Certain studies also show that mortality in women undergoing mechanical ventilation is significantly higher than that in men. Additionally, females are predisposed to certain cardiac electrophysiological risks, including QTc prolongation. In this study, we assessed the impact of hyperoxia in male and female mice (C57BL/6J) at age 8–10 weeks. On completion of either hyperoxia or normoxia exposures, physical, hemodynamic, biochemical, functional, electrophysiological, and molecular assessments were conducted. Hyperoxia-exposed mice lost a significant amount of body mass, compared with normoxia controls, in both sexes. However, while both genders developed brady-arrhythmia after hyperoxia exposure, female mice exhibited significantly reduced heart rates compared with males, with significantly elevated RR intervals. Additionally, 50% mortality was observed in females, whereas no mortality was reported in males. Furthermore, unlike in male mice, we observed no hypertrophy upon hyperoxia exposure in female mice. We reported that both hyperoxia-treated male and female mice exhibit significant hyperdynamic left ventricular ejection fraction, which is marked by % ejection fraction > 70 compared with the normoxia controls. We also noted significant reductions in stroke volume and cardiac output in both mice with hyperoxia. Surface ECG also demonstrated that hyperoxia exposure significantly augments RR, PR, QRS, QTc, and JT intervals in both sexes. Molecular analysis of left ventricular tissue demonstrated dysregulation of potassium ion channels in hyperoxia-treated males and females. In summary, we determined that sex differences are present with 72 hr hyperoxia exposure.     

Natural Prolongation: A Living Myth in the Regime of the Continental Shelf?

Since the 1969 North Sea Continental Shelf cases, natural prolongation has been a semisacred expression that characterizes the continental shelf. This article examines whether this notion is still viable. A comparative study of its current use in the U.N. Convention on the Law of the Sea against its original use reveals misunderstandings in three key aspects: entitlement, delineation, and delimitation of the continental shelf. This article concludes that natural prolongation no longer constitutes a pivot in the legal understanding of the continental shelf.     

The important but weakening maize yield benefit of grain filling prolongation in the US Midwest

A better understanding of recent crop yield trends is necessary for improving the yield and maintaining food security. Several possible mechanisms have been investigated recently in order to explain the steady growth in maize yield over the US Corn‐Belt, but a substantial fraction of the increasing trend remains elusive. In this study, trends in grain filling period (GFP) were identified and their relations with maize yield increase were further analyzed. Using satellite data from 2000 to 2015, an average lengthening of GFP of 0.37 days per year was found over the region, which probably results from variety renewal. Statistical analysis suggests that longer GFP accounted for roughly one‐quarter (23%) of the yield increase trend by promoting kernel dry matter accumulation, yet had less yield benefit in hotter counties. Both official survey data and crop model simulations estimated a similar contribution of GFP trend to yield. If growing degree days that determines the GFP continues to prolong at the current rate for the next 50 years, yield reduction will be lessened with 25% and 18% longer GFP under Representative Concentration Pathway 2.6 (RCP 2.6) and RCP 6.0, respectively. However, this level of progress is insufficient to offset yield losses in future climates, because drought and heat stress during the GFP will become more prevalent and severe. This study highlights the need to devise multiple effective adaptation strategies to withstand the upcoming challenges in food security.