Cationic Vitamin E-TPGS Mixed Micelles of Berberine to Neutralize Doxorubicin-Induced Cardiotoxicity via Amelioration of Mitochondrial Dysfunction and Impeding Apoptosis.

Anthracycline antibiotics, namely, doxorubicin (DOX) and daunorubicin, are among the most widely used anticancer therapies, yet are notoriously associated with severe myocardial damage due to oxidative stress and mitochondrial damage. Studies have indicated the strong pharmacological properties of Berberine (Brb) alkaloid, predominantly mediated via mitochondrial functions and nuclear networks. Despite the recent emphasis on Brb in clinical cardioprotective studies, pharmaceutical limitations hamper its clinical use. A nanoformulation for Brb was developed (mMic), incorporating a cationic lipid, oleylamine (OA), into the TPGS-mixed corona of PEGylated-phosphatidylethanolamine (PEG-PE) micelles. Cationic TPGS/PEG-PE mMic with superior Brb loading and stability markedly enhanced both intracellular and mitochondria-tropic Brb activities in cardiovascular muscle cells. Sub-lethal doses of Brb via cationic OA/TPGS mMic, as a DOX co-treatment, resulted in significant mitochondrial apoptosis suppression. In combination with an intense DOX challenge (up to ~50 µM), mitochondria-protective Brb-OA/TPGS mMic showed a significant 24 h recovery of cell viability (p ≤ 0.05-0.01). Mechanistically, the significant relative reduction in apoptotic caspase-9 and elevation of antiapoptotic Bcl-2 seem to mediate the cardioprotective role of Brb-OA/TPGS mMic against DOX. Our report aims to demonstrate the great potential of cationic OA/TPGS-mMic to selectively enhance the protective mitohormetic effect of Brb to mitigate DOX cardiotoxicity.

Efficient Ex Vivo Screening of Agents Targeting Thrombospondin1-Induced Vascular Dysfunction Using a Digital Multiwire Myograph System.

Homeostasis of vascular tone is intricately and delicately maintained systemically and locally, by autonomic nerves and hormones in the blood and by intimal vasoactive substances, respectively. The balance can be acutely or chronically interrupted secondary to many alterations, especially under pathological conditions. Excessive matricellular glycoprotein thrombospondin 1 (TSP1) levels in circulation have been found to play an important role in ischemia-reperfusion injuries of different organs, by acutely suppressing vasorelaxation and chronically remodeling vascular bed. Our laboratory has been interested in identifying new drug moieties, which can selectively and effectively counteract TSP1-induced vascular dysfunction, in order to address associated clinical complications. Preliminary studies using computational docking and molecular models revealed potential drug candidates for further evaluation via vascular functional bioassay to prove the antagonism using an ex vivo vascular model. Herein, we described an efficient screening method for the identification of active drug candidates, by adapting a multiwire myograph system to perform a protocol with different treatments, in the presence of pathological levels of TSP1. We discussed the promising pharmacological evaluation results and suggested suitable modification for versatile applications. We also described the necessity of pre-determination of optimal resting tension to obtain the maximal response, if the experimental test model is different from those with determined optimal resting tension.

Novel Pharmaceutical Strategy for Selective Abrogation of TSP1-Induced Vascular Dysfunction by Decoy Recombinant CD47 Soluble Receptor in Prophylaxis and Treatment Models.

Elevated thrombospondin 1 (TSP1) is a prevalent factor, via cognate receptor CD47, in the pathogenesis of cardiovascular conditions, including ischemia-reperfusion injury (IRI) and pulmonary arterial hypertension (PAH). Moreover, TSP1/CD47 interaction has been found to be associated with platelet hyperaggregability and impaired nitric oxide response, exacerbating progression in IRI and PAH. Pathological TSP1 in circulation arises as a target of our novel therapeutic approach. Our "proof-of-concept" pharmacological strategy relies on recombinant human CD47 peptide (rh-CD47p) as a decoy receptor protein (DRP) to specifically bind TSP1 and neutralize TSP1-impaired vasorelaxation, strongly implicated in IRI and PAH. The binding of rh-CD47p and TSP1 was first verified as the primary mechanism via Western blotting and further quantified with modified ELISA, which also revealed a linear molar dose-dependent interaction. Ex vivo, pretreatment protocol with rh-CD47p (rh-CD47p added prior to TSP1 incubation) demonstrated a prophylactic effect against TSP1-impairment of endothelium-dependent vasodilation. Post-treatment set-up (TSP1 incubation prior to rh-CD47p addition), mimicking pre-existing excessive TSP1 in PAH, reversed TSP1-inhibited vasodilation back to control level. Dose titration identified an effective molar dose range (approx. ≥1:3 of tTSP1:rh-CD47p) for prevention of/recovery from TSP1-induced vascular dysfunction. Our results indicate the great potential for proposed novel decoy rh-CD47p-therapy to abrogate TSP1-associated cardiovascular complications, such as PAH.

Liposomal Delivery of Cyclocreatine Impairs Cancer Cell Bioenergetics Mediating Apoptosis.

Creatine kinase (CK) enzyme overexpression has been suggested to play a role in the process of tumorigenesis and metastasis. Cyclocreatine (CCR) is a substrate analog of creatine kinase (CK), where its phosphorylated form is a poor phosphate donor in comparison with native bioenergetic molecule, creatine phosphate (Cr-P). The compound CCR has been shown to markedly inhibit the growth of a broad spectrum of cancers, both in vitro and in vivo. Intracellularly, CCR is phosphorylated by CK to yield a synthetic phosphagen [(N-phosphorylcyclocreatine (CCR ~P)], with thermodynamic and kinetic properties distinct from those of creatine phosphate (Cr-P). Distinct inhibition of tumor growth and metastasis has been attributed to CCR accumulation as CCR ~P in tumor cells, especially in those expressing a high level of CK protein, with minimal adverse effects. Unfortunately, the clinical use of CCR against malignancies is quite limited due to its amphoteric nature, which accounts for most of its extremely low membrane permeability, as well as limited oral bioavailability (BA) and poor systemic pharmacokinetics (PK).Our current work describes the encapsulation of CCR , utilizing freeze and thaw vesicles (FTV )-composed mostly of saturated PC, DOPE, and Chol-into stealth™ liposomes , postcoated with 4.5 M% PEG-PE. Following physicochemical characterization, in vitro release and cellular uptake kinetics confirmed efficient delivery of liposomal CCR (CCR-Lip), leading to intracellular accumulation of its CC-P metabolic product. Successful delivery of CCR to cancer cell effectively depleted low energetic cancer cells of ATP significantly mediating myc-induced metabolic changes. CCR-Lip showed significant antimetastatic and anticancer effectiveness against both MCF-7 and PC-3 human carcinoma models (p < 0.05-0.01), with 4- to 6-fold lower IC50 values vs. closest drug control. Such shift in bioenergetics was coupled via AMPK and phospho-p53 to the mitochondrial apoptosis effector Bak , thus inducing a cell-intrinsic mechanism to counteract uncontrolled neoplastic proliferation, in target cancer cells. Our novel liposomal delivery system of the CCR substrate analog demonstrated strong inhibition of malignant cell bioenergetics, leading to significant antineoplastic and proapoptotic actions, against different cancers.