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.

Conjugation of amiodarone to a novel cardiomyocyte cell penetrating peptide for potential targeted delivery to the heart.

Modern medicine has developed a myriad of therapeutic drugs against a wide range of human diseases leading to increased life expectancy and better quality of life for millions of people. Despite the undeniable benefit of medical advancements in pharmaceutical technology, many of the most effective drugs currently in use have serious limitations such as off target side effects resulting in systemic toxicity. New generations of specialized drug constructs will enhance targeted therapeutic efficacy of existing and new drugs leading to safer and more effective treatment options for a variety of human ailments. As one of the most efficient drugs known for the treatment of cardiac arrhythmia, Amiodarone presents the same conundrum of serious systemic side effects associated with long term treatment. In this article we present the synthesis of a next-generation prodrug construct of amiodarone for the purpose of advanced targeting of cardiac arrhythmias by delivering the drug to cardiomyocytes using a novel cardiac targeting peptide, a cardiomyocyte-specific cell penetrating peptide. Our in vivo studies in guinea pigs indicate that cardiac targeting peptide-amiodarone conjugate is able to have similar effects on calcium handling as amiodarone at 1/15th the total molar dose of amiodarone. Further studies are warranted in animal models of atrial fibrillation to show efficacy of this conjugate.

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.