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

Yurko, Ray; Islam, Md. Kamrul; Weber, Beth; Salama, Guy; Zahid, Maliha

doi:10.3389/fchem.2023.1220573

Cited by 6 publications

(10 citation statements)

References 33 publications

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“…Amiodarone–thiol was then purified by preparative C-18 RP-HPLC purification on a Waters Delta Prep 4000 chromatography system and then lyophilized. The purified amiodarone–thiol was then reacted with pNpys-CTP in ammonium acetate buffered solution at pH 4 for 2 h. Preparative C-18 RP-HPLC purification on a Waters Delta Prep 4000 chromatography system was followed by lyophilization to a dry powder [ 32 ]. Analytical C-18 RP-HPLC characterization of Amiodarone-SS-CTP on a Waters Alliance chromatography system was performed followed by mass analysis on a Bruker UltraFlextreme MALDI TOF/TOF mass spectrometer (Framingham, MA, USA) to confirm the expected mass and purity of the final product.…”

Section: Methodsmentioning

confidence: 99%

“…Analytical C-18 RP-HPLC characterization of Amiodarone-SS-CTP on a Waters Alliance chromatography system was performed followed by mass analysis on a Bruker UltraFlextreme MALDI TOF/TOF mass spectrometer (Framingham, MA, USA) to confirm the expected mass and purity of the final product. Please see the publication by R. Yurko and colleagues for the step-by-step detailed synthesis protocol [ 32 ].…”

Section: Methodsmentioning

confidence: 99%

“…All p -values are two-tailed unpaired t -tests between each treatment group and the controls. (Panel ( b ) is reproduced by permission from Frontiers in Chemistry [ 32 ]).…”

Section: Figurementioning

confidence: 99%

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Cardiomyocyte-Targeting Peptide to Deliver Amiodarone

et al. 2023

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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-1β, 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.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Cardiomyocyte-Targeting Peptide to Deliver Amiodarone

et al. 2023

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“…This linker was chosen due to its covalent nature and high likelihood of remaining stable in serum, as well as being broken down in the reducing intracellular environment, leading to release of cargo miRNA106a from its vector CTP, allowing it to bind to calcium calmodulin kinase Iiδ mRNA marking it for degradation in the RISC complex. Incubation of human left ventricular myocytes with fluorescently labeled CTP-miRNA106a conjugate led to robust uptake by the cells [50]. This conjugate was selectively taken up by human cardiomyocyte cell line and not by HEK293 cells [50].…”

Section: Cardiac-targeting Peptide For Therapeutic Purposesmentioning

confidence: 98%

Cardiac-Targeting Peptide: From Discovery to Applications

Sahagun,

Zahid

2023

Biomolecules

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Despite significant strides in prevention, diagnosis, and treatment, cardiovascular diseases remain the number one cause of mortality in the United States, with rates climbing at an alarming rate in the developing world. Targeted delivery of therapeutics to the heart has been a lofty goal to achieve with strategies ranging from direct intra-cardiac or intra-pericardial delivery, intra-coronary infusion, to adenoviral, lentiviral, and adeno-associated viral vectors which have preference, if not complete cardio-selectivity, for cardiac tissue. Cell-penetrating peptides (CPP) are 5–30-amino-acid-long peptides that are able to breach cell membrane barriers while carrying cargoes up to several times their size, in an intact functional form. Identified nearly three decades ago, the first of these CPPs came from the HIV coat protein transactivator of transcription. Although a highly efficient CPP, its clinical utility is limited by its robust ability to cross any cell membrane barrier, including crossing the blood–brain barrier and transducing neuronal tissue non-specifically. Several strategies have been utilized to identify cell- or tissue-specific CPPs, one of which is phage display. Using this latter technique, we identified a cardiomyocyte-targeting peptide (CTP) more than a decade ago, a finding that has been corroborated by several independent labs across the world that have utilized CTP for a myriad of different purposes in pre-clinical animal models. The goal of this publication is to provide a comprehensive review of the identification, validation, and application of CTP, and outline its potential in diagnostic and therapeutic applications especially in the field of targeted RNA interference.

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“…CTP appears to primarily transduce the heart and is not taken up by the lungs, spleen, or other muscle subtypes, and does not cross the blood–brain barrier [ 15 ]. The cell-specific transduction ability of CTP has been validated several times by independent laboratories as being specific to cardiomyocytes and has been shown to be effective at delivering a variety of cargoes, including photosensitizers [ 19 ], exosomes [ 18 ], miRNA for the treatment of heart failure [ 20 ], and amiodarone [ 21 ] at significantly reduced doses, suggesting a potential for reduction in off-target side effects while retaining amiodarone functionality [ 22 ]. A recent publication summarized all of this accumulated literature on CTP [ 13 ].…”

Section: Introductionmentioning

confidence: 99%

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

Sahagun,

Lopuszynski,

Feldman

et al. 2024

Pharmaceutics

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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.

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

Cited by 6 publications

References 33 publications

Cardiomyocyte-Targeting Peptide to Deliver Amiodarone

Cardiomyocyte-Targeting Peptide to Deliver Amiodarone

Cardiac-Targeting Peptide: From Discovery to Applications

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

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