Forced degradation of therapeutic proteins

The scope of this paper is to review approaches used for forced degradation (synonym, stress testing) of therapeutic proteins. Forced degradation studies play a central role in the development of therapeutic proteins, for example, for candidate selection, molecule characterization, formulation development, assay development, and comparability studies. Typical stress methods are addressed within this review, such as exposure to elevated temperatures, freeze-thawing, mechanical stress, oxidation, light, as well as various materials and devices used in the clinics during final administration. Stability testing is briefly described as far as relevant to the discussion of forced degradation studies. Whereas stability-testing requirements are defined in regulatory guidelines, standard procedures for forced degradation of therapeutic proteins are largely unavailable, except for photostability. Possible selection criteria to identify appropriate stress conditions and recommendations for setting up forced degradation studies for the different phases of development of therapeutic proteins are presented.     

Taste masked lipid pellets with enhanced release of hydrophobic active ingredient

Solid lipid extrusion is a suitable technique to produce oral dosage forms with improved taste properties. The design of a lipid formulation for poorly water soluble drugs is a challenge because of the poor dissolution and potential bioavailability problems. In this study, solid lipid extrusion at room temperature was applied for the formulation development of the BCS Class II drug NXP 1210. Powdered hard fat (Witocan(?) 42/44 mikrofein), glycerol distearate (Precirol(?) ato 5) and glycerol trimyristate (Dynasan(?) 114) were investigated as lipid binders. Different amounts of polyvinylalcohol (PVA)-polyethyleneglycol (PEG)-graft copolymer (Kollicoat(?) IR) and crospovidone (Polyplasdone(?) Xl-10) were scrutinized as solubilizers. The dissolution profiles depicted a short lag time (about 2min) and then fast and complete dissolution of NXP 1210 by increasing the amount of crospovidone. The initial release was more delayed with an increased amount of PVA-PEG-graft copolymer. Dissolution rate could also be influenced by changing the lipid binder from pure hard fat into a mixture of hard fat, glycerol distearate and glycerol trimyristate. The formulations are feasible for taste-masked granules or pellets containing poorly soluble drugs.     

Decision-making in Polydrug Amphetamine-type Stimulant Users: an fMRI Study

Qualitative poor decision-making and associated altered neuronal activation patterns have been described for the users of several drugs, amongst others for stimulants like amphetamine and MDMA. Deficits in decision-making might be caused by an augmented attraction to short-term rewarding properties despite negative long-term consequences, leading to rigid stimulus–response patterns. In the present imaging study, we investigated decision-making and associated neuronal activation in three groups differing in their exposure to amphetamine and MDMA. An established paradigm on risky choices was used to evaluate decision-making performance and corresponding functional magnet resonance imaging (fMRI) activation. Subjects could choose between a low-risk control gamble and an experimental gamble, which always differed in the probability of winning or losing, as well as the magnitudes of monetary gain or loss. Experienced users (EU), users with low exposure to stimulants and drug-naive controls, did not differ from each other in behavioral performance. In accordance with our hypotheses, the anticipation of reward led to an activation of primarily the frontal cortex and the striatum in low-exposure users and drug-naive controls. In contrast, frontal and parietal activation was observed in all groups when the actual outcome of an experimental gamble was presented. EU displayed more activation compared to both control groups when there was a high probability of winning. The study at hand supports the hypothesis that neuronal activation patterns might even differ between drug users and healthy controls when no behavioral deficits are apparent. In EU, the probability of the occurrence of an event has more influence on neuronal activation than on the actual magnitude of reinforcing properties of this event.     

Effect of filtration by activated charcoal on the toxicological activity of cigarette mainstream smoke from experimental cigarettes

Activated charcoal (AC) filtration reportedly decreases the yields of smoke vapor phase constituents including some identified as human carcinogens and respiratory irritants. Non-clinical studies including chemical smoke analysis, in vitro cytotoxicity and mutagenicity (bacterial and mammalian cells), and in vivo subchronic rat inhalation studies were carried out using machine smoking at ISO conditions with lit-end research cigarettes containing AC filters. The objective was to assess whether AC filter technology would alter the established toxicity profile of mainstream smoke by increasing or decreasing any known toxicological properties, or elicit new ones. The reduced yield of vapor phase irritants from AC filter cigarettes correlated with markedly decreased in vitro cytotoxicity and in vivo morphology of the nose and lower respiratory tract. Increased yields of particulate phase constituents (e.g. polycyclic aromatic hydrocarbons) in AC filtered smoke were noted in comparison to controls in some studies. The in vitro bacterial mutagenicity of AC filtered smoke particulate preparations was occasionally increased over control levels. Laryngeal epithelial thickness was increased in some rats inhaling AC filtered smoke in comparison to controls, an effect perhaps related to higher inspiratory flow. When tested under more intense Massachusetts Department of Public Health smoking conditions, AC filter associated reductions in vapor phase constituent yields were smaller than those seen with ISO conditions, but the effect on in vitro cytotoxicity remained.     

Impact of intracoronary reinfusion of bone marrow-derived mononuclear progenitor cells on cardiopulmonary exercise capacity in patients with chronic postinfarction heart failure

Introduction

Intracoronary infusion of bone marrow-derived progenitor cells (BMC) in patients with chronic ischaemic heart failure (CHF) is associated with improvement in left ventricular ejection fraction (LVEF), reduction of NT-proBNP levels and improved prognosis. However, effects of this therapy on cardiopulmonary exercise capacity have not been investigated separately so far.

Patients and methods

One hundred and fifty-four patients with ischaemic heart failure (mean LVEF 40.3 ± 10.9 %, NT-proBNP 1,103 ± 1,436 pg/ml) underwent cardiopulmonary exercise capacity testing (CPX) before and 3 months after intracoronary infusion of autologous BMC. Thirty patients with a potential bias on the CPX course as concomitant coronary intervention, bypass surgery, new onset of arrhythmias or implantation of cardiac resynchronization devices were excluded from further analysis.

Results

The remaining 124 patients showed an increase in exercise time and peak workload by 16.8 and 6 %. Peak oxygen uptake and oxygen uptake efficiency slope also improved by 2.9 and 12.9 %, whereas other parameters like peak oxygen pulse and the slope of minute ventilation versus CO₂ elimination remained unchanged. Analysis of patients with poor, moderate and conserved CPX results prior to cell therapy documented that patients in tertiles with lowest initial exercise capacity showed the largest improvements in CPX after therapy. The differences in response to cell therapy were detectable in all investigated CPX parameters and became significant for exercise time, peak oxygen uptake and peak oxygen pulse.

Summary

These findings indicate that intracoronary BMC therapy improves exercise capacity in CHF patients with more advanced heart failure.     

Severe hyperphosphatemia in a patient with chronic kidney disease and multiple myeloma–to strengthen the case toward renal replacement therapy?

We report a patient with multiple myeloma and chronic kidney disease who presented with severe hyperphosphatemia in the outpatient clinic without any related symptoms. Initial differential diagnosis: Tumor lysis syndrome or chronic kidney disease. Further work‐up revealed pseudohyperphosphatemia. In general, treatment is not necessary if the true phosphate level is within the reference range and the patient is asymptomatic.     

Fundamentals and Potential of Magnetic Particle Imaging

Cardiovascular interventions are standard treatment for numerous cardiovascular conditions and require high fidelity imaging tools to accurately visualize both vessels and interventional devices. Currently, digital subtraction angiography (DSA) is the standard method for peripheral arterial angiography. Magnetic particle imaging (MPI) is a new imaging modality, free of ionizing radiation, that utilizes static and oscillating magnetic fields to provide high temporal resolution, sub-millimeter spatial resolution images and high sensitivity. Superparamagnetic iron oxide nanoparticles (SPIOs) are used as tracers in MPI and signals are based on non-linear magnetization characteristics of those SPIOs. Regarding the magnetic moment of used tracers in MPI imaging is much faster in MPI, compared to imaging in CT and MRI. This makes MPI also very attractive for cardiovascular imaging and cardiovascular interventions. First in vivo visualization of a beating mouse heart demonstrated the feasibility of the visualization of the cardiovascular system by MPI. Different scanner designs and acquisition methods have already emerged addressing the requirements of cardiovascular interventions. Early studies have demonstrated MPI as an interesting and promising cardiovascular imaging modality. Technical improvement in hardware MPI imaging systems are currently being addressed in ongoing research which will facilitate former image acquisition with higher resolution in larger animals and/or human.     

Effect of duplex drugs linking 2′-deoxy-5-fluorouridine (5-FdU) with 3′-C-ethynylcytidine (ECyd) on hepatoblastoma cell lines

Purpose

Duplex drugs are promising anticancer agents. After in vivo cleavage into active nucleoside analogues, they exert their anti-tumour activity with reduced toxicity and side effects. Here we evaluated the impact of two duplex drugs on the viability of hepatoblastoma (HB) cells lines and their toxicity against human fibroblasts.

Methods

The duplex drugs 2′-deoxy-5-fluorouridylyl-(3′-5′)- 3′-C-ethynylcytidine (5-FdU(3′-5′)ECyd) and 3′-C-ethynylcytidinylyl-(5′→1-O)-2-O-octadecyl-sn-glycerylyl-(3′-Ο→5′)-2′-deoxy-5-fluorouridine (ECyd-lipid-5-FdU) were analysed in two HB cell lines (HUH6, HepT1) and fibroblasts by MTT assay. The treatment potential was compared to the single substances 2′-deoxy-5-fluorourindine (5-FdU), 3′-C-ethynylycytidine (ECyd) and an equimolar mixture of both. Cell cycle analyses were performed using flow cytometry after 7-AAD staining.

Results

Both duplex drugs achieve a potent cytotoxic effect at low μM concentrations, which was more pronounced than the mixture of ECyd + 5-FdU. Further, both substances exert toxicity on fibroblasts of tumour samples, with less toxicity in foreskin fibroblasts cultures. Cell cycle analyses revealed a shift towards apoptotic cells for both drugs in HB cells.

Conclusion

5-FdU(3′-5′)ECyd and ECyd-lipid-5-FdU exert a highly potent anti-tumoural effect on HB cells and might therefore be a treatment option in HB. Pharmacological formulations of both duplex drugs have to be evaluated in vivo to reduce possible side effects.