The Structural Dispersity of Oligoethylene Glycol-Containing Polymer Brushes Determines Their Interfacial Properties.

Ought to their bioinert properties and facile synthesis, poly[(oligoethylene glycol)methacrylate]s (POEGMAs) have been raised as attractive alternatives to poly(ethylene glycols) (PEGs) in an array of (bio)material applications, especially when they are applied as polymer brush coatings. However, commercially available OEG-methacrylate (macro)monomers feature a broad distribution of OEG lengths, thus generating structurally polydisperse POEGMAs when polymerized through reversible deactivation radical polymerization. Here, we demonstrate that the interfacial physicochemical properties of POEGMA brushes are significantly affected by their structural dispersity, i.e., the degree of heterogeneity in the length of side OEG segments. POEGMA brushes synthesized from discrete (macro)monomers obtained through chromatographic purification of commercial mixtures show increased hydration and reduced adhesion when compared to their structurally polydisperse analogues. The observed alteration of interfacial properties is directly linked to the presence of monodisperse OEG side chains, which hamper intramolecular and intermolecular hydrophobic interactions while simultaneously promoting the association of water molecules. These phenomena provide structurally homogeneous POEGMA brushes with a more lubricious and protein repellent character with respect to their heterogeneous counterparts. More generally, in contrast to what has been assumed until now, the properties of POEGMA brushes cannot be anticipated while ruling out the effect of dispersity by (macro)monomer feeds. Simultaneously, side chain dispersity of POEGMAs emerges as a critical parameter for determining the interfacial characteristics of brushes.

Sphingosine is involved in PAPTP-induced death of pancreas cancer cells by interfering with mitochondrial functions.

Pancreas ductal adenocarcinoma belongs to the most common cancers, but also to the tumors with the poorest prognosis. Here, we pharmacologically targeted a mitochondrial potassium channel, namely mitochondrial Kv1.3, and investigated the role of sphingolipids and mutated Kirsten Rat Sarcoma Virus (KRAS) in Kv1.3-mediated cell death. We demonstrate that inhibition of Kv1.3 using the Kv1.3-inhibitor PAPTP results in an increase of sphingosine and superoxide in membranes and/or membranes associated with mitochondria, which is enhanced by KRAS mutation. The effect of PAPTP on sphingosine and mitochondrial superoxide formation as well as cell death is prevented by sh-RNA-mediated downregulation of Kv1.3. Induction of sphingosine in human pancreas cancer cells by PAPTP is mediated by activation of sphingosine-1-phosphate phosphatase and prevented by an inhibitor of sphingosine-1-phosphate phosphatase. A rapid depolarization of isolated mitochondria is triggered by binding of sphingosine to cardiolipin, which is neutralized by addition of exogenous cardiolipin. The significance of these findings is indicated by treatment of mutated KRAS-harboring metastasized pancreas cancer with PAPTP in combination with ABC294640, a blocker of sphingosine kinases. This treatment results in increased formation of sphingosine and death of pancreas cancer cells in vitro and, most importantly, prolongs in vivo survival of mice challenged with metastatic pancreas cancer. KEY MESSAGES: Pancreatic ductal adenocarcinoma (PDAC) is a common tumor with poor prognosis. The mitochondrial Kv1.3 ion channel blocker induced mitochondrial sphingosine. Sphingosine binds to cardiolipin thereby mediating mitochondrial depolarization. Sphingosine is formed by a PAPTP-mediated activation of S1P-Phosphatase. Inhibition of sphingosine-consumption amplifies PAPTP effects on PDAC in vivo.

Effect of Time From Onset of Major Depressive Disorder on the Therapeutic Response to Esmethadone (REL-1017).

Objective: The relationship between the duration of major depressive disorder (MDD) and therapeutic response to standard antidepressant treatment (SAT) is unknown. N-methyl-D-aspartate receptor uncompetitive antagonists are emerging drugs for MDD. We investigated whether the antidepressant effect of esmethadone (REL-1017) could be related to the duration of depression.Methods: We analyzed data from a Phase 2a study of adjunctive treatment with esmethadone in MDD patients (DSM-5) with inadequate response to ongoing SAT (May 2018-August 2019). Patients were randomized to treatment with esmethadone 25 mg, esmethadone 50 mg, or placebo for 7 days, followed by an observation period (Days 7-14). Duration of depression was derived from 2 measures: (1) time from onset (TFO), calculated as the difference in years between age at trial enrollment and age at the onset of the first major depressive episode (MDE), and (2) TFO index, calculated by computing the years of illness duration (number of years from the beginning of MDD), divided by age and multiplied by 100. First, bivariate correlations between TFO and change from baseline (CFB) were calculated by Spearman ρ. Linear mixed-model analyses were also conducted.Results: A total of 62 patients participated in the trial. The median values of time from MDD onset for the 62 patients were 11 years (absolute value) and 22% (percentage of life-years). Duration of depression was significantly correlated with Montgomery-Asberg Depression Rating Scale (MADRS) CFB on Day 14, even when controlling for the effect of current depression severity (MADRS baseline). In the linear mixed-model analyses, we found a significant effect of duration on reduction in MADRS score from T0 to subsequent assessments (P < .05). Number of previous MDEs and effect of esmethadone 50 mg when compared to 25 mg were not significant.Conclusion: Esmethadone 25 and 50 mg were more effective in reducing MADRS scores in patients with shorter time from first MDE onset.Trial Registration: ClinicalTrials.gov identifier: NCT03051256.

The mitochondrial ATP-dependent potassium channel (mitoKATP) controls skeletal muscle structure and function.

MitoKATP is a channel of the inner mitochondrial membrane that controls mitochondrial K+ influx according to ATP availability. Recently, the genes encoding the pore-forming (MITOK) and the regulatory ATP-sensitive (MITOSUR) subunits of mitoKATP were identified, allowing the genetic manipulation of the channel. Here, we analyzed the role of mitoKATP in determining skeletal muscle structure and activity. Mitok-/- muscles were characterized by mitochondrial cristae remodeling and defective oxidative metabolism, with consequent impairment of exercise performance and altered response to damaging muscle contractions. On the other hand, constitutive mitochondrial K+ influx by MITOK overexpression in the skeletal muscle triggered overt mitochondrial dysfunction and energy default, increased protein polyubiquitination, aberrant autophagy flux, and induction of a stress response program. MITOK overexpressing muscles were therefore severely atrophic. Thus, the proper modulation of mitoKATP activity is required for the maintenance of skeletal muscle homeostasis and function.

Efficient SARS-CoV-2 infection antagonization by rhACE2 ectodomain multimerized onto the Avidin-Nucleic-Acid-NanoASsembly.

Nanodecoy systems based on analogues of viral cellular receptors assembled onto fluid lipid-based membranes of nano/extravescicles are potential new tools to complement classic therapeutic or preventive antiviral approaches. The need for lipid-based membranes for transmembrane receptor anchorage may pose technical challenges along industrial translation, calling for alternative geometries for receptor multimerization. Here we developed a semisynthetic self-assembling SARS-CoV-2 nanodecoy by multimerizing the biotin labelled virus cell receptor -ACE2- ectodomain onto a poly-avidin nanoparticle (NP) based on the Avidin-Nucleic-Acid-NanoASsembly-ANANAS. The ability of the assembly to prevent SARS-CoV-2 infection in human lung cells and the affinity of the ACE2:viral receptor-binding domain (RBD) interaction were measured at different ACE2:NP ratios. At ACE2:NP = 30, 90 % SARS-CoV-2 infection inhibition at ACE2 nanomolar concentration was registered on both Wuhan and Omicron variants, with ten-fold higher potency than the monomeric protein. Lower and higher ACE2 densities were less efficient suggesting that functional recognition between multi-ligand NPs and multi-receptor virus surfaces requires optimal geometrical relationships. In vivo studies in mice showed that the biodistribution and safety profiles of the nanodecoy are potentially suitable for preventing viral infection upon nasal instillation. Viral receptor multimerization using ANANAS is a convenient process which, in principle, could be rapidly adapted to counteract also other viral infections.

Drug-Drug Interaction Studies of Esmethadone (REL-1017) Involving CYP3A4- and CYP2D6-Mediated Metabolism.

BACKGROUND AND OBJECTIVE: Esmethadone (dextromethadone; d-methadone; S-methadone (+)-methadone; REL-1017) is the opioid inactive dextro-isomer of racemic methadone. Esmethadone is a low potency N-methyl-D-aspartate (NMDA) receptor channel blocker with higher affinity for GluN2D subtypes. Esmethadone showed robust, rapid, and sustained antidepressant effects in patients with major depressive disorder (MDD) with inadequate response to ongoing serotonergic antidepressant treatment. METHODS: Here we described the results of in vitro and phase 1 clinical trials aimed at investigating the esmethadone metabolism and possible drug-drug interactions. RESULTS: Esmethadone is primarily metabolized to EDDP (2-ethylene-1,5-dimethyl-3,3-diphenylpyrrolidine) by multiple enzymes, including CYP3A4/5 and CYP2B6. In vitro studies showed that esmethadone inhibits CYP2D6 with IC50 of 9.6 µM and is an inducer of CYP3A4/5. The clinical relevance of the inhibition of CYP2D6 and the induction of CYP3A4 were investigated by co-administering esmethadone and dextromethorphan (a substrate for CYP2D6) or midazolam (a substrate for CYP3A4) in healthy volunteers. The administration of esmethadone at the dosage of 75 mg (which is the loading dose administered to patients in MDD clinical trials) significantly increased the exposure (AUC) of both dextromethorphan and its metabolite dextrorphan by 2.71 and 3.11-fold, respectively. Esmethadone did not modify the pharmacokinetic profile of midazolam, while it increased Cmax and AUC of its metabolite 1'-hydroxymidazolam by 2.4- and 3.8-fold, respectively. A second study evaluated the effect of the CYP3A4 inhibitor cobicistat on the pharmacokinetics of esmethadone. Cobicistat slightly increase (+32%) the total exposure (AUC0-inf) of esmethadone. CONCLUSIONS: In summary, esmethadone demonstrated a negligible effect on CYP3A4 induction and its metabolism was not meaningfully affected by strong CYP3A4 inhibitors while it increased exposure of CYP2D6-metabolized drugs.

The novel uncompetitive NMDA receptor antagonist esmethadone (REL-1017) has no meaningful abuse potential in recreational drug users.

Esmethadone (REL-1017) is the opioid-inactive dextro-isomer of methadone and a low-affinity, low-potency uncompetitive NMDA receptor antagonist. In a Phase 2, randomized, double-blind, placebo-controlled trial, esmethadone showed rapid, robust, and sustained antidepressant effects. Two studies were conducted to evaluate the abuse potential of esmethadone. Each study utilized a randomized, double-blind, active-, and placebo-controlled crossover design to assess esmethadone compared with oxycodone (Oxycodone Study) or ketamine (Ketamine Study) in healthy recreational drug users. Esmethadone 25 mg (proposed therapeutic daily dose), 75 mg (loading dose), and 150 mg (Maximum Tolerated Dose) were evaluated in each study. Positive controls were oral oxycodone 40 mg and intravenous ketamine 0.5 mg/kg infused over 40 min. The Ketamine study included oral dextromethorphan 300 mg as an exploratory comparator. The primary endpoint was maximum effect (Emax) for Drug Liking, assessed using a bipolar 100-point visual analog scale (VAS). A total of 47 and 51 participants completed the Oxycodone Study and the Ketamine Study, respectively (Completer Population). In both studies, esmethadone doses ranging from therapeutic (25 mg) to 6 times therapeutic (150 mg) had a meaningful and statistically significantly (p < 0.001) lower Drug Liking VAS Emax compared with the positive control. Results were consistent for all secondary endpoints in both studies. In both studies, all doses of esmethadone were statistically equivalent to placebo on Drug Liking VAS Emax (p < 0.05). In the Ketamine Study, Drug Liking VAS Emax scores for esmethadone at all tested doses were significantly lower vs. dextromethorphan (p < 0.05) (exploratory endpoint). These studies indicate no meaningful abuse potential for esmethadone at all tested doses.

DA7R: A 7-Letter Zip Code to Target PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer, and is among the most aggressive and still incurable cancers. Innovative and successful therapeutic strategies are extremely needed. Peptides represent a versatile and promising tool to achieve tumor targeting, thanks to their ability to recognize specific target proteins (over)expressed on the surface of cancer cells. A7R is one such peptide, binding neuropilin-1 (NRP-1) and VEGFR2. Since PDAC expresses these receptors, the aim of this study was to test if A7R-drug conjugates could represent a PDAC-targeting strategy. PAPTP, a promising mitochondria-targeted anticancer compound, was selected as the cargo for this proof-of-concept study. Derivatives were designed as prodrugs, using a bioreversible linker to connect PAPTP to the peptide. Both the retro-inverso (DA7R) and the head-to-tail cyclic (cA7R) protease-resistant analogs of A7R were tested, and a tetraethylene glycol chain was introduced to improve solubility. Uptake of a fluorescent DA7R conjugate, as well as of the PAPTP-DA7R derivative into PDAC cell lines was found to be related to the expression levels of NRP-1 and VEGFR2. Conjugation of DA7R to therapeutically active compounds or nanovehicles might allow PDAC-targeted drug delivery, improving the efficacy of the therapy and reducing off-target effects.

Esmethadone-HCl (REL-1017): a promising rapid antidepressant.

This review article presents select recent studies that form the basis for the development of esmethadone into a potential new drug. Esmethadone is a promising member of the pharmacological class of uncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonists that have shown efficacy for major depressive disorder (MDD) and other diseases and disorders, such as Alzheimer's dementia and pseudobulbar affect. The other drugs in the novel class of NMDAR antagonists with therapeutic uses that are discussed for comparative purposes in this review are esketamine, ketamine, dextromethorphan, and memantine. We present in silico, in vitro, in vivo, and clinical data for esmethadone and other uncompetitive NMDAR antagonists that may advance our understanding of the role of these receptors in neural plasticity in health and disease. The efficacy of NMDAR antagonists as rapid antidepressants may advance our understanding of the neurobiology of MDD and other neuropsychiatric diseases and disorders.

Defective excitation-contraction coupling and mitochondrial respiration precede mitochondrial Ca2+ accumulation in spinobulbar muscular atrophy skeletal muscle.

Polyglutamine expansion in the androgen receptor (AR) causes spinobulbar muscular atrophy (SBMA). Skeletal muscle is a primary site of toxicity; however, the current understanding of the early pathological processes that occur and how they unfold during disease progression remains limited. Using transgenic and knock-in mice and patient-derived muscle biopsies, we show that SBMA mice in the presymptomatic stage develop a respiratory defect matching defective expression of genes involved in excitation-contraction coupling (ECC), altered contraction dynamics, and increased fatigue. These processes are followed by stimulus-dependent accumulation of calcium into mitochondria and structural disorganization of the muscle triads. Deregulation of expression of ECC genes is concomitant with sexual maturity and androgen raise in the serum. Consistent with the androgen-dependent nature of these alterations, surgical castration and AR silencing alleviate the early and late pathological processes. These observations show that ECC deregulation and defective mitochondrial respiration are early but reversible events followed by altered muscle force, calcium dyshomeostasis, and dismantling of triad structure.

Subanalysis of Subjective Cognitive Measures From a Phase 2, Double-Blind, Randomized Trial of REL-1017 in Patients With Major Depressive Disorder.

Objective: Improvement of cognitive function in patients with major depressive disorder (MDD) is an important treatment outcome. REL-1017 (esmethadone HCl) is a novel N-methyl-d-aspartate receptor (NMDAR) channel blocker and a potentially rapidly acting antidepressant. The objective of this study was to define the effects of REL-1017 on subjective cognitive measures in patients with MDD.Methods: Post hoc analysis was conducted of subjective cognitive measures from the Montgomery-Asberg Depression Rating Scale (MADRS) and the Symptoms of Depression Questionnaire (SDQ) from a randomized, double-blind, placebo-controlled, Phase 2a study. The study, designed to assess the safety, tolerability, and efficacy of 2 dosages (25 mg and 50 mg) of REL-1017 as an adjunctive treatment in patients with MDD unresponsive to standard antidepressants, included 62 patients. We analyzed subjective cognitive measures derived from the MADRS and SDQ scales at baseline and up to day 14, 7 days after the last dose of study drug. We developed 2 composite indexes that included subjective cognitive measures selected from the MADRS and SDQ.Results: The subanalysis of single measures and the 2 composite indexes derived from the MADRS and SDQ measures showed clinically meaningful and statistically significant improvements in cognitive function (P < .05).Conclusions: In a Phase 2a clinical trial, REL-1017 improved subjective measures of cognitive impairment, in addition to improving total MADRS and SDQ scores. These results need to be confirmed in larger and longer studies in MDD that include objective measures of cognitive function. Phase 3 studies of REL-1017 for MDD are currently underway.Clinical Trials Registration: ClinicalTrials.gov identifier: NCT03051256.

Pharmacological targeting of the mitochondrial calcium-dependent potassium channel KCa3.1 triggers cell death and reduces tumor growth and metastasis in vivo.

Ion channels are non-conventional, druggable oncological targets. The intermediate-conductance calcium-dependent potassium channel (KCa3.1) is highly expressed in the plasma membrane and in the inner mitochondrial membrane (mitoKCa3.1) of various cancer cell lines. The role mitoKCa3.1 plays in cancer cells is still undefined. Here we report the synthesis and characterization of two mitochondria-targeted novel derivatives of a high-affinity KCa3.1 antagonist, TRAM-34, which retain the ability to block channel activity. The effects of these drugs were tested in melanoma, pancreatic ductal adenocarcinoma and breast cancer lines, as well as in vivo in two orthotopic models. We show that the mitochondria-targeted TRAM-34 derivatives induce release of mitochondrial reactive oxygen species, rapid depolarization of the mitochondrial membrane, fragmentation of the mitochondrial network. They trigger cancer cell death with an EC50 in the µM range, depending on channel expression. In contrast, inhibition of the plasma membrane KCa3.1 by membrane-impermeant Maurotoxin is without effect, indicating a specific role of mitoKCa3.1 in determining cell fate. At sub-lethal concentrations, pharmacological targeting of mitoKCa3.1 significantly reduced cancer cell migration by enhancing production of mitochondrial reactive oxygen species and nuclear factor-κB (NF-κB) activation, and by downregulating expression of Bcl-2 Nineteen kD-Interacting Protein (BNIP-3) and of Rho GTPase CDC-42. This signaling cascade finally leads to cytoskeletal reorganization and impaired migration. Overexpression of BNIP-3 or pharmacological modulation of NF-κB and CDC-42 prevented the migration-reducing effect of mitoTRAM-34. In orthotopic models of melanoma and pancreatic ductal adenocarcinoma, the tumors at sacrifice were 60% smaller in treated versus untreated animals. Metastasis of melanoma cells to lymph nodes was also drastically reduced. No signs of toxicity were observed. In summary, our results identify mitochondrial KCa3.1 as an unexpected player in cancer cell migration and show that its pharmacological targeting is efficient against both tumor growth and metastatic spread in vivo.

Esmethadone (REL-1017) and Other Uncompetitive NMDAR Channel Blockers May Improve Mood Disorders via Modulation of Synaptic Kinase-Mediated Signaling.

This article presents a mechanism of action hypothesis to explain the rapid antidepressant effects of esmethadone (REL-1017) and other uncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonists and presents a corresponding mechanism of disease hypothesis for major depressive disorder (MDD). Esmethadone and other uncompetitive NMDAR antagonists may restore physiological neural plasticity in animal models of depressive-like behavior and in patients with MDD via preferential tonic block of pathologically hyperactive GluN2D subtypes. Tonic Ca2+ currents via GluN2D subtypes regulate the homeostatic availability of synaptic proteins. MDD and depressive behaviors may be determined by reduced homeostatic availability of synaptic proteins, due to upregulated tonic Ca2+ currents through GluN2D subtypes. The preferential activity of low-potency NMDAR antagonists for GluN2D subtypes may explain their rapid antidepressant effects in the absence of dissociative side effects.

Pharmacological Comparative Characterization of REL-1017 (Esmethadone-HCl) and Other NMDAR Channel Blockers in Human Heterodimeric N-Methyl-D-Aspartate Receptors.

Excessive Ca2+ currents via N-methyl-D-aspartate receptors (NMDARs) have been implicated in many disorders. Uncompetitive NMDAR channel blockers are an emerging class of drugs in clinical use for major depressive disorder (MDD) and other neuropsychiatric diseases. The pharmacological characterization of uncompetitive NMDAR blockers in clinical use may improve our understanding of NMDAR function in physiology and pathology. REL-1017 (esmethadone-HCl), a novel uncompetitive NMDAR channel blocker in Phase 3 trials for the treatment of MDD, was characterized together with dextromethorphan, memantine, (±)-ketamine, and MK-801 in cell lines over-expressing NMDAR subtypes using fluorometric imaging plate reader (FLIPR), automated patch-clamp, and manual patch-clamp electrophysiology. In the absence of Mg2+, NMDAR subtypes NR1-2D were most sensitive to low, sub-µM glutamate concentrations in FLIPR experiments. FLIPR Ca2+ determination demonstrated low µM affinity of REL-1017 at NMDARs with minimal subtype preference. In automated and manual patch-clamp electrophysiological experiments, REL-1017 exhibited preference for the NR1-2D NMDAR subtype in the presence of 1 mM Mg2+ and 1 µM L-glutamate. Tau off and trapping characteristics were similar for (±)-ketamine and REL-1017. Results of radioligand binding assays in rat cortical neurons correlated with the estimated affinities obtained in FLIPR assays and in automated and manual patch-clamp assays. In silico studies of NMDARs in closed and open conformation indicate that REL-1017 has a higher preference for docking and undocking the open-channel conformation compared to ketamine. In conclusion, the pharmacological characteristics of REL-1017 at NMDARs, including relatively low affinity at the NMDAR, NR1-2D subtype preference in the presence of 1 mM Mg2+, tau off and degree of trapping similar to (±)-ketamine, and preferential docking and undocking of the open NMDAR, could all be important variables for understanding the rapid-onset antidepressant effects of REL-1017 without psychotomimetic side effects.

The N-Methyl-D-Aspartate Receptor Blocker REL-1017 (Esmethadone) Reduces Calcium Influx Induced by Glutamate, Quinolinic Acid, and Gentamicin.

REL-1017 (esmethadone) is a novel N-methyl-D-aspartate receptor (NMDAR) antagonist and promising rapid antidepressant candidate. Using fluorometric imaging plate reader (FLIPR) assays, we studied the effects of quinolinic acid (QA) and gentamicin, with or without L-glutamate and REL-1017, on intracellular calcium ([Ca2+]in) in recombinant cell lines expressing human GluN1-GluN2A, GluN1-GluN2B, GluN1-GluN2C, and GluN1-GluN2D NMDAR subtypes. There were no effects of QA on [Ca2+]in in cells expressing GluN1-GluN2C subtypes. QA acted as a low-potency, subtype-selective, NMDAR partial agonist in GluN1-GluN2A, GluN1-GluN2B, and GluN1-GluN2D subtypes. REL-1017 reduced [Ca2+]in induced by QA. In cells expressing the GluN1-GluN2D subtype, QA acted as an agonist in the presence of 0.04 µM L-glutamate and as an antagonist in the presence of 0.2 µM L-glutamate. REL-1017 reduced [Ca2+]in induced by L-glutamate alone and with QA in all cell lines. In the absence of L-glutamate, gentamicin had no effect. Gentamicin was a positive modulator for GluN1-GluN2B subtypes at 10 µM L-glutamate, for GluN1-GluN2A at 0.2 µM L-glutamate, and for GluN1-GluN2A, GluN1-GluN2B, and GluN1-GluN2D at 0.04 µM L-glutamate. No significant changes were observed with GluN1-GluN2C NMDARs. REL-1017 reduced [Ca2+]in induced by the addition of L-glutamate in all NMDAR cell lines in the presence or absence of gentamicin. In conclusion, REL-1017 reduced [Ca2+]in induced by L-glutamate alone and when increased by QA and gentamicin. REL-1017 may protect cells from excessive calcium entry via NMDARs hyperactivated by endogenous and exogenous molecules.

Inhibition of a Mitochondrial Potassium Channel in Combination with Gemcitabine and Abraxane Drastically Reduces Pancreatic Ductal Adenocarcinoma in an Immunocompetent Orthotopic Murine Model.

Pancreas ductal adenocarcinoma (PDAC) is one the most aggressive cancers and associated with very high mortality, requiring the development of novel treatments. The mitochondrial voltage-gated potassium channel, Kv1.3 is emerging as an attractive oncologic target but its function in PDAC is unknown. Here, we evaluated the tissue expression of Kv1.3 in resected PDAC from 55 patients using immunohistochemistry (IHC) and show that all tumors expressed Kv1.3 with 60% of tumor specimens having high Kv1.3 expression. In Pan02 cells, the recently developed mitochondria-targeted Kv1.3 inhibitors PCARBTP and PAPTP strongly reduced cell survival in vitro. In an orthotopic pancreas tumor model (Pan02 cells injected into C57BL/6 mice) in immune-competent mice, injection of PAPTP or PCARBTP resulted in tumor reductions of 87% and 70%, respectively. When combined with clinically used Gemcitabine plus Abraxane (albumin-bound paclitaxel), reduction reached 95% and 80% without resultant organ toxicity. Drug-mediated tumor cell death occurred through the p38-MAPK pathway, loss of mitochondrial membrane potential, and oxidative stress. Resistant Pan02 clones to PCARBTP escaped cell death through upregulation of the antioxidant system. In contrast, tumor cells did not develop resistance to PAPTP. Our data show that Kv1.3 is highly expressed in resected human PDAC and the use of novel mitochondrial Kv1.3 inhibitors combined with cytotoxic chemotherapies might be a novel, effective treatment for PDAC.

Mitochondrial Kv1.3 Channels as Target for Treatment of Multiple Myeloma.

Despite several new developments in the treatment of multiple myeloma, all available therapies are only palliative without curative potential and all patients ultimately relapse. Thus, novel therapeutic options are urgently required to prolong survival of or to even cure myeloma. Here, we show that multiple myeloma cells express the potassium channel Kv1.3 in their mitochondria. The mitochondrial Kv1.3 inhibitors PAPTP and PCARBTP are efficient against two tested human multiple myeloma cell lines (L-363 and RPMI-8226) and against ex vivo cultured, patient-derived myeloma cells, while healthy bone marrow cells are spared from toxicity. Cell death after treatment with PAPTP and PCARBTP occurs via the mitochondrial apoptotic pathway. In addition, we identify up-regulation of the multidrug resistance pump MDR-1 as the main potential resistance mechanism. Combination with ABT-199 (venetoclax), an inhibitor of Bcl2, has a synergistic effect, suggesting that mitochondrial Kv1.3 inhibitors could potentially be used as combination partner to venetoclax, even in the treatment of t(11;14) negative multiple myeloma, which represent the major part of cases and are rather resistant to venetoclax alone. We thus identify mitochondrial Kv1.3 channels as druggable targets against multiple myeloma.

Pharmacological modulation of Kv1.3 potassium channel selectively triggers pathological B lymphocyte apoptosis in vivo in a genetic CLL model.

BACKGROUND: Ion channels are emerging as promising oncological targets. The potassium channels Kv1.3 and IKCa are highly expressed in the plasma membrane and mitochondria of human chronic lymphocytic leukemia (CLL) cells, compared to healthy lymphocytes. In vitro, inhibition of mitoKv1.3 by PAPTP was shown to kill ex vivo primary human CLL cells, while targeting IKCa with TRAM-34 decreased CLL cell proliferation. METHODS: Here we evaluated the effect of the above drugs in CLL cells from ibrutinib-resistant patients and in combination with Venetoclax, two drugs used in the clinical practice. The effects of the drugs were tested also in the Eµ-TCL1 genetic CLL murine model, characterized by a lympho-proliferative disease reminiscent of aggressive human CLL. Eµ-TCL1 mice showing overt disease state were treated with intraperitoneal injections of non-toxic 5 nmol/g PAPTP or 10 nmol/g TRAM-34 once a day and the number and percentage of pathological B cells (CD19+CD5+) in different, pathologically relevant body districts were determined. RESULTS: We show that Kv1.3 expression correlates with sensitivity of the human and mouse neoplastic cells to PAPTP. Primary CLL cells from ibrutinib-resistant patients could be killed with PAPTP and this drug enhanced the effect of Venetoclax, by acting on mitoKv1.3 of the inner mitochondrial membrane and triggering rapid mitochondrial changes and cytochrome c release. In vivo, after 2 week- therapy of Eµ-TCL1 mice harboring distinct CLL clones, leukemia burden was reduced by more than 85%: the number and percentage of CLL B cells fall in the spleen and peritoneal cavity and in the peripheral blood, without signs of toxicity. Notably, CLL infiltration into liver and spleen and splenomegaly were also drastically reduced upon PAPTP treatment. In contrast, TRAM-34 did not exert any beneficial effect when administered in vivo to Eµ-TCL1 mice at non-toxic concentration. CONCLUSION: Altogether, by comparing vehicle versus compound effect in different Eµ-TCL1 animals bearing unique clones similarly to CLL patients, we conclude that PAPTP significantly reduced leukemia burden in CLL-relevant districts, even in animals with advanced stage of the disease. Our results thus identify PAPTP as a very promising drug for CLL treatment, even for the chemoresistant forms of the disease.