Phospholamban antisense oligonucleotides improve cardiac function in murine cardiomyopathy

Heart failure (HF) is a major cause of morbidity and mortality worldwide, highlighting an urgent need for novel treatment options, despite recent improvements. Aberrant Ca(2+) handling is a key feature of HF pathophysiology. Restoring the Ca(2+) regulating machinery is an attractive therapeutic strategy supported by genetic and pharmacological proof of concept studies. Here, we study antisense oligonucleotides (ASOs) as a therapeutic modality, interfering with the PLN/SERCA2a interaction by targeting Pln mRNA for downregulation in the heart of murine HF models. Mice harboring the PLN R14del pathogenic variant recapitulate the human dilated cardiomyopathy (DCM) phenotype; subcutaneous administration of PLN-ASO prevents PLN protein aggregation, cardiac dysfunction, and leads to a 3-fold increase in survival rate. In another genetic DCM mouse model, unrelated to PLN (Cspr3/Mlp(−/−)), PLN-ASO also reverses the HF phenotype. Finally, in rats with myocardial infarction, PLN-ASO treatment prevents progression of left ventricular dilatation and improves left ventricular contractility. Thus, our data establish that antisense inhibition of PLN is an effective strategy in preclinical models of genetic cardiomyopathy as well as ischemia driven HF

Quantification of unbound concentration of ticagrelor in plasma as a proof of mechanism biomarker of the reversal agent, MEDI2452.

Ticagrelor, a P2Y12 antagonist, is approved for prevention of thromboembolic events. MEDI2452 is a potential reversal agent for ticagrelor and ticagrelor active metabolite (TAM). The total plasma exposure of ticagrelor and TAM in patients are roughly 0.5-1 and 0.2-0.5 μmol/L, respectively. Both have similar high potency vs. P2Y12 (Ki 2 nmol/L) but are plasma protein-bound to 99.8% and only the 0.2% free fraction is able to inhibit the P2Y12 receptor. Thus, for unbound concentration measurements to be a proof of mechanism biomarker for MEDI2452 a very high sensitivity is required. Using established techniques as equilibrium dialysis and LC-MS/MS, made it possible to evaluate the efficacy of the reversal agent by measuring reduction of unbound concentration of ticagrelor in the presence of MEDI2452. With challenges such as ultra-low concentrations, small sample volumes, recovery issues and adsorption to plastic we managed to develop a highly sensitive assay for determining unbound concentration levels of ticagrelor and TAM in plasma with a quantification limit of 30 pmol/L and 45 pmol/L, respectively. With this method we were able to detect close to a 100-fold MEDI2452 mediated reduction in the unbound concentration of both ticagrelor and TAM. The assay provided proof of mechanism as MEDI2452 concentration- and dose-dependently eliminated unbound concentration of ticagrelor and reversed its antiplatelet activity in preclinical models and will support future development of MEDI2452

Accurate measurement of endogenous adenosine in human blood.

Accurate determination of in vivo circulating concentrations of extracellular adenosine in blood samples is challenging due to the rapid formation and rapid clearance of adenosine in blood. A blood collection protocol was developed based on direct sampling of venous blood into, and instant mixing with, a STOP solution developed to conserve in vivo adenosine concentrations by completely preventing both its formation and clearance in collected blood. Stable isotope labeled AMP and adenosine spiked into blood ex vivo were used in combination with mass spectrometry to evaluate conservation of adenosine and prevention of its formation. A number of approved drugs, including the P2Y12 antagonist ticagrelor, have been described to increase extracellular adenosine. This may contribute to its clinical profile, highlighting the importance of accurate measurement of in vivo adenosine concentrations.A high sensitive ultra performance liquid chromatography-tandem- mass spectrometry (UPLC-tandem-MS) analytical method for plasma adenosine was developed and validated with a lower limit of quantification of 2 nmol/L. The method demonstrated plasma adenosine stability during sample processing and analytical method performance relevant to human blood samples. The final STOP solution proved able to conserve exogenous adenosine and to prevent adenosine formation from exogenous AMP added in vitro to human blood over 15 minutes. The mean endogenous adenosine concentration in plasma prepared from venous blood collected from 10 healthy volunteers was 13 ± 7 nmol/L. Finally, the method was used to demonstrate the previously described concentration-dependent ability of ticagrelor to conserve extracellular adenosine at clinically relevant exposures. In conclusion, we report an optimized sampling protocol and a validated analytical method for accurate measurement of in vivo circulating adenosine concentrations in human blood, suitable for use in clinical trials

Rapid Production of Human VEGF-A following Intradermal Injection of Modified VEGF-A mRNA Demonstrated by Cutaneous Microdialysis in the Rabbit and Pig In Vivo

Chemically modified mRNA is a novel, highly efficient, biocompatible modality for therapeutic protein expression that may overcome the challenges and safety concerns with current gene therapy strategies. We explored the efficiency of intradermally injected modified VEGF-A165 mRNA (VEGF-A mRNA) formulated in a biocompatible citrate/saline buffer to locally produce human VEGF-A165 protein. Rabbits (n=4) and minipigs (n=3) were implanted with subcutaneous microdialysis probes close to the injection sites and interstitial-fluid samples and skin biopsies were analysed for production of VEGF-A protein over time for up to 8 hours. Three to 4 hours after the intradermal injection of VEGF-A mRNA, detectable levels of human VEGF-A protein were seen in the microdialysis eluates in both species. In the pig, the VEGF-A concentrations increased dose-dependently reaching a maximum 6 hours after dosing (62.7±28.4, 357.6±240.6, and 746.3±210.2 pg/mL following injection of 24, 120, and 600 μg VEGF-A mRNA, respectively). Likewise, in tissue biopsies harvested at study end (8 hours after VEGF-A mRNA injection), the content of VEGF-A protein increased dose-dependently. In contrast, VEGF-A protein was not detected in eluates originating from sites injected with citrate/saline vehicle. It is concluded that intradermal injection of VEGF-A mRNA is associated with a rapid and local production of VEGF-A protein. Considering the pro-angiogenic effect of VEGF-A, VEGF-A mRNA may hold promise for regenerative treatment of patients with diabetic wounds and ischemic cardiovascular disease

Recovery of ticagrelor in presence of MEDI2452 using different protocols to prepare the plasma retentate.

<p>No formic acid (●, n = 4), 0.17% formic acid (final concentration) added at time of protein precipitation (□, n = 2) and 0.17% formic acid (final concentration) added 10 minutes prior to protein precipitation (▲, n = 8). Human PRP was pre-incubated in vitro with ticagrelor (1 μmol/L) for 60 minutes followed by addition of MEDI2452 (0.25, 0.5, 0.75, 1 and 2 μmol/L) and 30 minutes further incubation. Data expressed as mean ± SD.</p

Ticagrelor fraction unbound (<i>fu</i>) measured in plasma samples with different concentrations of ticagrelor.

<p>Human plasma sample pre-incubated in vitro for 60 minutes. Data expressed as mean ± SD (n = 3). Statistical differences were calculated using one-way ANOVA test (p = 0.249). Tukey’s multiple comparison test was used to look at differences between the lowest, 0.025 μmol/L, and highest, 10 μmol/L, concentration groups (p = 0.845).</p

Comparison of recovery using LoBind tubes and generic Safe-Lock tubes.

<p>Comparison of recovery using LoBind tubes and generic Safe-Lock tubes.</p

Reproducibility of ticagrelor fraction unbound (<i>fu</i>) at different plasma volumes in the dialysis.

<p>Human plasma samples were incubated with ticagrelor, 1 μmol/L (theoretical <i>fu</i> value ~ 0.2%). Data expressed as mean ± SD (n = 5). Statistical differences were calculated using one-way ANOVA test (p = 0.619). Tukey’s multiple comparison test were used to look at differences between the volume groups, 100 μL vs. 130 μL (p = 0.692), 100 μL vs. 150 μL (p = 0.649).</p

Representative LC-MS/MS chromatograms of calibration samples in buffer.

<p>(A) Ticagrelor, 0.4 nmol/L (B) TAM, 0.6 nmol/L and (C) internal standard.</p