Toxicity Studies of Cardiac-Targeting Peptide Reveal a Robust Safety Profile.

Targeted delivery of therapeutics specifically to cardiomyocytes would open up new frontiers for common conditions like heart failure. Our prior work using a phage display methodology identified a 12-amino-acid-long peptide that selectively targets cardiomyocytes after an intravenous injection in as little as 5 min and was hence termed a cardiac-targeting peptide (CTP: APHLSSQYSRT). CTP has been used to deliver imaging agents, small drug molecules, photosensitizing nanoparticles, exosomes, and even miRNA to cardiomyocytes. As a natural extension to the development of CTP as a clinically viable cardiac vector, we now present toxicity studies performed with the peptide. In vitro viability studies were performed in a human left ventricular myocyte cell line with 10 µM of Cyanine-5.5-labeled CTP (CTP-Cy5.5). In vitro ion channel profiles were completed for CTP followed by extensive studies in stably transfected cell lines for several GPCR-coupled receptors. Positive data for GPCR-coupled receptors were interrogated further with RT-qPCRs performed on mouse heart tissue. In vivo studies consisted of pre- and post-blood pressure monitoring acutely after a single CTP (10 mg/Kg) injection. Further in vivo toxicity studies consisted of injecting CTP (150 µg/Kg) in 60, 6-week-old, wild-type CD1, male/female mice (1:1), with cohorts of mice euthanized on days 0, 1, 2, 7, and 14 with inhalational CO2, followed by blood collection via cardiac puncture, complete blood count analysis, metabolic profiling, and finally, liver, renal, and thyroid studies. Lastly, mouse cardiac MRI was performed immediately before and after CTP (150 µg/Kg) injection to assess changes in cardiac size or function. Human left ventricular cardiomyocytes showed no decrease in viability after a 30 min incubation with CTP-Cy5.5. No significant activation or inhibition of any of seventy-eight protein channels was observed other than OPRM1 and COX2 at the highest tested concentration, neither of which were expressed in mouse heart tissue as assessed using RT-qPCR. CTP (10 mg/Kg) injections led to no change in blood pressure. Blood counts and chemistries showed no evidence of significant hematological, hepatic, or renal toxicities. Lastly, there was no difference in cardiac function, size, or mass acutely in response to CTP injections. Our studies with CTP showed no activation or inhibition of GPCR-associated receptors in vitro. We found no signals indicative of toxicity in vivo. Most importantly, cardiac functions remained unchanged acutely in response to CTP uptake. Further studies using good laboratory practices are needed with prolonged, chronic administration of CTP conjugated to a specific cargo of choice before human studies can be contemplated.

Cardiomyocyte-Targeting Peptide to Deliver Amiodarone.

BACKGROUND: Amiodarone is underutilized due to significant off-target toxicities. We hypothesized that targeted delivery to the heart would lead to the lowering of the dose by utilizing a cardiomyocyte-targeting peptide (CTP), a cell-penetrating peptide identified by our prior phage display work. METHODS: CTP was synthesized thiolated at the N-terminus, conjugated to amiodarone via Schiff base chemistry, HPLC purified, and confirmed with MALDI/TOF. The stability of the conjugate was assessed using serial HPLCs. Guinea pigs (GP) were injected intraperitoneally daily with vehicle (7 days), amiodarone (7 days; 80 mg/kg), CTP-amiodarone (5 days; 26.3 mg/kg), or CTP (5 days; 17.8 mg/kg), after which the GPs were euthanized, and the hearts were excised and perfused on a Langendorff apparatus with Tyrode's solution and blebbistatin (5 µM) to minimize the contractions. Voltage (RH237) and Ca2+-indicator dye (Rhod-2/AM) were injected, and fluorescence from the epicardium split and was captured by two cameras at 570-595 nm for the cytosolic Ca2+ and 610-750 nm wavelengths for the voltage. Subsequently, the hearts were paced at 250 ms with programmed stimulation to measure the changes in the conduction velocities (CV), action potential duration (APD), and Ca2+ transient durations at 90% recovery (CaTD90). mRNA was extracted from all hearts, and RNA sequencing was performed with results compared to the control hearts. RESULTS: The CTP-amiodarone remained stable for up to 21 days at 37 °C. At ~1/15th of the dose of amiodarone, the CTP-amiodarone decreased the CV in hearts significantly compared to the control GPs (0.92 ± 0.05 vs. 1.00 ± 0.03 ms, p = 0.0007), equivalent to amiodarone alone (0.87 ± 0.08 ms, p = 0.0003). Amiodarone increased the APD (192 ± 5 ms vs. 175 ± 8 ms for vehicle, p = 0.0025), while CTP-amiodarone decreased it significantly (157 ± 16 ms, p = 0.0136), similar to CTP alone (155 ± 13 ms, p = 0.0039). Both amiodarone and CTP-amiodarone significantly decreased the calcium transients compared to the controls. CTP-amiodarone and CTP decreased the CaTD90 to an extent greater than amiodarone alone (p < 0.001). RNA-seq showed that CTP alone increased the expression of DHPR and SERCA2a, while it decreased the expression of the proinflammatory genes, NF-kappa B, TNF-α, IL-1ß, and IL-6. CONCLUSIONS: Our data suggest that CTP can deliver amiodarone to cardiomyocytes at ~1/15th the total molar dose of the amiodarone needed to produce a comparable slowing of CVs. The ability of CTP to decrease the AP durations and CaTD90 may be related to its increase in the expression of Ca-handling genes, which merits further study.

Cardiomyocyte Targeting Peptide to Deliver Amiodarone.

Background: Amiodarone is underutilized due to significant off-target toxicities. We hypothesized that targeted delivery to the heart would lead to lowering of dose by utilizing a cardiomyocyte targeting peptide (CTP), a cell penetrating peptide identified by our prior phage display work. Methods: CTP was synthesized thiolated at the N-terminus, conjugated to amiodarone via Schiff base chemistry, HPLC purified and confirmed with MALDI/TOF. Stability of the conjugate was assessed using serial HPLCs. Guinea pigs (GP) were injected intraperitoneally daily with vehicle (7 days), amiodarone (7 days; 80mg/Kg), CTP-amiodarone (5 days;26.3mg/Kg), or CTP (5 days; 17.8mg/Kg), after which GPs were euthanized, hearts excised, perfused on a Langendorff apparatus with Tyrode's solution and blebbistatin (5µM) to minimize contractions. Voltage (RH237) and Ca 2+ -indicator dye (Rhod-2/AM) were injected, fluorescence from the epicardium split and focused on two cameras capturing at 570-595nm for cytosolic Ca 2+ and 610-750nm wavelengths for voltage. Subsequently, hearts were paced at 250ms with programmed stimulation to measure changes in conduction velocities (CV), action potential duration (APD) and Ca 2+ transient durations at 90% recovery (CaTD 90 ). mRNA was extracted from all hearts and RNA sequencing performed with results compared to control hearts. Results: CTP-amiodarone remained stable for up to 21 days at 37°C. At ∼1/15 th of the dose of amiodarone, CTP-amiodarone decreased CV in hearts significantly compared to control GPs (0.92±0.05 vs. 1.00±0.03m/s, p=0.0007), equivalent to amiodarone alone (0.87±0.08ms, p=0.0003). Amiodarone increased APD (192±5ms vs. 175±8ms for vehicle, p=0.0025), while CTP-amiodarone decreased it significantly (157±16ms, p=0.0136) similar to CTP alone (155±13ms, p=0.0039). Both amiodarone and CTP-amiodarone significantly decreased calcium transients compared to controls. CTP-amiodarone and CTP decreased CaTD 90 to an extent greater than amiodarone alone (p<0.001). RNA-seq showed that CTP alone increased the expression of DHPR and SERCA2a, while decreasing expression of proinflammatory genes NF-kappa B, TNF-α, IL-1ß, and IL-6. Conclusions: Our data suggests that CTP can deliver amiodarone to cardiomyocytes at ∼1/15 th the total molar dose of amiodarone needed to produce comparable slowing of CVs. The ability of CTP to decrease AP durations and CaTD 90 may be related to its increase in expression of Ca-handling genes, and merits further study.

Prognosticators of All-Cause Mortality in Patients With Heart Failure With Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF) represents ∼50% of all cases of congestive heart failure (CHF) with prevalence expected to increase with aging of the population. We performed an observational study of all patients admitted to 3 hospitals in the ExcelaHealth care system, Greensburg, PA, with a primary diagnosis of HFpEF heart failure exacerbation between January 2014 and January 2017. Demographic data, laboratory results, and echocardiograms performed closest to index hospitalization were collected. A total of 487 patients were admitted with a primary diagnosis of CHF exacerbation and HFpEF, with a mean age of 80.5 years (±10.9), 62% women and predominantly Caucasian (98.8%). Over a median follow-up of 21.7 months, 246 patients died with an all-cause mortality rate of 51.3%. Receiver operator curves were generated for multiple continuous variables to identify optimal cut-off values Kaplan-Meir survival curves were then generated. Clinical factors were tested by univariate Cox regression modeling, with significant factors entered into a step-wise multivariate model. Our modeling identified age>80 years, serum albumin level<3.2 g/dl, N-terminal pro-brain natriuretic peptide (NT-proBNP) >5,000 pg/mL and medial E/e'≥20 as significant, independent predictors of all-cause mortality (p-value <0.0001). Surprisingly, lack of a diagnosis of hypertension was associated with significantly increased mortality risk. In a community-based sample of HFpEF patients, we identified multiple factors that were strong, independent predictors of all-cause mortality that can be easily applied in a clinical setting.