Publications by authors named "Henrik Franzyk"

111 Publications

Targeting Toxins toward Tumors.

Molecules 2021 Feb 27;26(5). Epub 2021 Feb 27.

Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark.

Many cancer diseases, e.g., prostate cancer and lung cancer, develop very slowly. Common chemotherapeutics like vincristine, vinblastine and taxol target cancer cells in their proliferating states. In slowly developing cancer diseases only a minor part of the malignant cells will be in a proliferative state, and consequently these drugs will exert a concomitant damage on rapidly proliferating benign tissue as well. A number of toxins possess an ability to kill cells in all states independently of whether they are benign or malignant. Such toxins can only be used as chemotherapeutics if they can be targeted selectively against the tumors. Examples of such toxins are mertansine, calicheamicins and thapsigargins, which all kill cells at low micromolar or nanomolar concentrations. Advanced prodrug concepts enabling targeting of these toxins to cancer tissue comprise antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT), lectin-directed enzyme-activated prodrug therapy (LEAPT), and antibody-drug conjugated therapy (ADC), which will be discussed in the present review. The review also includes recent examples of protease-targeting chimera (PROTAC) for knockdown of receptors essential for development of tumors. In addition, targeting of toxins relying on tumor-overexpressed enzymes with unique substrate specificity will be mentioned.
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http://dx.doi.org/10.3390/molecules26051292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7956858PMC
February 2021

Optimizing the Intracellular Delivery of Therapeutic Anti-inflammatory TNF-α siRNA to Activated Macrophages Using Lipidoid-Polymer Hybrid Nanoparticles.

Front Bioeng Biotechnol 2020 14;8:601155. Epub 2021 Jan 14.

Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

RNA interference (RNAi) has an unprecedented potential as a therapeutic strategy for reversibly silencing the expression of any gene. Therapeutic delivery of the RNAi mediator, i.e., small interfering RNA (siRNA), can be used to address diseases characterized by gene overexpression, for example inflammatory conditions like chronic obstructive pulmonary disease (COPD). Macrophages play a key role in COPD pathogenesis and are recruited to the airways and lung parenchyma, where they release proinflammatory cytokines, e.g., tumor necrosis factor-alpha (TNF-α). Hence, targeting TNF-α with siRNA is a promising therapeutic approach for COPD management. However, a safe and effective delivery system is required for delivery of TNF-α siRNA into the cytosol of hard-to-transfect macrophages. The purpose of this study was to optimize the intracellular delivery of TNF-α siRNA to the lipopolysaccharide-activated murine macrophage cell line RAW 264.7 using lipidoid-polymer hybrid nanoparticles (LPNs) composed of the lipid-like transfection agent lipidoid 5 (L) and the biodegradable polymer poly (D,L-lactide-co-glycolide). Applying a quality-by-design approach, the influence of critical formulation variables, i.e., the L content and the L:siRNA ratio (w/w), on critical quality attributes (CQAs) was investigated systematically using risk assessment and design of experiments, followed by delineation of an optimal operating space (OOS). The CQAs were identified based on the quality target product profile and included size, polydispersity index, zeta potential, encapsulation efficiency and loading for achieving efficient and safe TNF-α gene silencing in activated RAW 264.7 cells. Formulations inducing efficient gene silencing and low cytotoxicity were identified, and the optimal formulations displayed L contents of 15 and 20% (w/w), respectively, and an L:siRNA weight ratio of 15:1. All tested formulations within the OOS mediated efficient and sequence-specific TNF-α gene silencing in RAW 264.7 cells at TNF-α-siRNA concentrations, which were significantly lower than the concentrations required of non-encapsulated TNF-α-siRNA, highlighting the benefit of the delivery system. The results also demonstrate that increasing the loading of siRNA into the delivery system does not necessarily imply enhanced gene silencing. This opens new avenues for further exploitation of LPNs as a robust platform technology for delivering TNF-α siRNA to macrophages, e.g., in the management of COPD.
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http://dx.doi.org/10.3389/fbioe.2020.601155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7841370PMC
January 2021

Alternating Cationic-Hydrophobic Peptide/Peptoid Hybrids: Influence of Hydrophobicity on Antibacterial Activity and Cell Selectivity.

ChemMedChem 2020 Dec 7;15(24):2544-2561. Epub 2020 Oct 7.

Department of Drug Design and Pharmacology, University of Copenhagen, Jagtvej 162, 2100, Copenhagen, Denmark.

The influence of hydrophobicity on antibacterial activity versus the effect on the viability of mammalian cells for peptide/peptoid hybrids was examined for oligomers based on the cationic Lys-like peptoid residue combined with each of 28 hydrophobic amino acids in an alternating sequence. Their relative hydrophobicity was correlated to activity against both Gram-negative and Gram-positive species, human red blood cells, and HepG2 cells. This identified hydrophobic side chains that confer potent antibacterial activity (e. g., MICs of 2-8 μg/mL against E. coli) and low toxicity toward mammalian cells (<10 % hemolysis at 400 μg/mL and IC >800 μg/mL for HepG2 viability). Most peptidomimetics retained activity against drug-resistant strains. These findings corroborate the hypothesis that for related peptidomimetics two hydrophobicity thresholds may be identified: i) it should exceed a certain level in order to confer antibacterial activity, and ii) there is an upper limit, beyond which cell selectivity is lost. It is envisioned that once identified for a given subclass of peptide-like antibacterials such thresholds can guide further optimisation.
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http://dx.doi.org/10.1002/cmdc.202000526DOI Listing
December 2020

Anti-inflammatory Activity of Lipidated Peptidomimetics Pam-(Lys-βNspe)-NH and Lau-(Lys-βNspe)-NH Against PMA-Induced Acute Inflammation.

Front Immunol 2020 28;11:2102. Epub 2020 Aug 28.

Faculty of Health and Medical Sciences, Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark.

Host Defense Peptides (HDPs) are key components of innate immunity that exert antimicrobial, antibiofilm, and immunomodulatory activities in all higher organisms. Synthetic peptidomimetic analogs were designed to retain the desirable pharmacological properties of HDPs while having improved stability toward enzymatic degradation, providing enhanced potential for therapeutic applications. Lipidated peptide/β-peptoid hybrids [e.g., Pam-(Lys-βNspe)-NH (PM1) and Lau-(Lys-βNspe)-NH (PM2)] are proteolytically stable HDP mimetics displaying anti-inflammatory activity and formyl peptide receptor 2 antagonism in human and mouse immune cells . Here PM1 and PM2 were investigated for their anti-inflammatory activity in a phorbol 12-myristate 13-acetate (PMA)-induced acute mouse ear inflammation model. Topical administration of PM1 or PM2 led to attenuated PMA-induced ear edema, reduced local production of the pro-inflammatory chemokines MCP-1 and CXCL-1 as well as the cytokine IL-6. In addition, diminished neutrophil infiltration into PMA-inflamed ear tissue and suppressed local release of reactive oxygen and nitrogen species were observed upon treatment. The obtained results show that these two peptidomimetics exhibit anti-inflammatory effects comparable to that of the non-steroidal anti-inflammatory drug indomethacin, and hence possess a potential for treatment of inflammatory skin conditions.
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http://dx.doi.org/10.3389/fimmu.2020.02102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7485003PMC
August 2020

Barbadin selectively modulates FPR2-mediated neutrophil functions independent of receptor endocytosis.

Biochim Biophys Acta Mol Cell Res 2020 12 8;1867(12):118849. Epub 2020 Sep 8.

Department of Rheumatology and Inflammation Research, University of Gothenburg, Gothenburg, Sweden. Electronic address:

FPR2, a member of the family of G protein-coupled receptors (GPCRs), mediates neutrophil migration, a response that has been linked to β-arrestin recruitment. β-Arrestin regulates GPCR endocytosis and can also elicit non-canonical receptor signaling. To determine the poorly understood role of β-arrestin in FPR2 endocytosis and in NADPH-oxidase activation in neutrophils, Barbadin was used as a research tool in this study. Barbadin has been shown to bind the clathrin adaptor protein (AP2) and thereby prevent β-arrestin/AP2 interaction and β-arrestin-mediated GPCR endocytosis. In agreement with this, AP2/β-arrestin interaction induced by an FPR2-specific agonist was inhibited by Barbadin. Unexpectedly, however, Barbadin did not inhibit FPR2 endocytosis, indicating that a mechanism independent of β-arrestin/AP2 interaction may sustain FPR2 endocytosis. This was confirmed by the fact, that FPR2 also underwent agonist-promoted endocytosis in β-arrestin deficient cells, albeit at a diminished level as compared to wild type cells. Dissection of the Barbadin effects on FPR2-mediated neutrophil functions including NADPH-oxidase activation mediated release of reactive oxygen species (ROS) and chemotaxis revealed that Barbadin had no effect on chemotactic migration whereas the release of ROS was potentiated/primed. The effect of Barbadin on ROS production was reversible, independent of β-arrestin recruitment, and similar to that induced by latrunculin A. Taken together, our data demonstrate that endocytic uptake of FPR2 occurs independently of β-arrestin, while Barbadin selectively augments FPR2-mediated ROS production independently of receptor endocytosis. Given that Barbadin binds to AP2 and prevents the AP2/β-arrestin interaction, our results indicate a role for AP2 in FPR2-mediated ROS release from neutrophils.
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http://dx.doi.org/10.1016/j.bbamcr.2020.118849DOI Listing
December 2020

Novel Cyclic Lipopeptide Antibiotics: Effects of Acyl Chain Length and Position.

Int J Mol Sci 2020 Aug 13;21(16). Epub 2020 Aug 13.

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark.

Multidrug-resistant bacteria are a global health problem. One of the last-resort antibiotics against Gram-negative bacteria is the cyclic lipopeptide colistin, displaying a flexible linker with a fatty acid moiety. The aim of the present project was to investigate the effect on antimicrobial activity of introducing fatty acid moieties of different lengths and in different positions in a cyclic peptide, S3(B), containing a flexible linker. The lipidated analogues of S3(B) were synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis. Following assembly of the linear peptide by Fmoc solid-phase peptide synthesis, on-resin head-to-tail cyclization and fatty acid acylation were performed. The antimicrobial activity was determined against the ESKAPE pathogens, , , , , and . Furthermore, hemolytic activity was determined against human erythrocytes. A total of 18 cyclic lipopeptides were synthesized and characterized. It was found that introduction of fatty acids in positions next to the flexible linker was more strongly linked to antimicrobial activity. The fatty acid length altered the overall hydrophobicity, which was the driving force for both high antimicrobial and hemolytic activity. Peptides became highly hemolytic when carbon-chain length exceeded 10 (i.e., C), overlapping with the optimum for antimicrobial activity (i.e., C-C). The most promising candidate (C) showed antimicrobial activity corresponding to that of S3(B), but with an improved hemolytic profile. Finally, (C) was further investigated in a time-kill experiment.
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http://dx.doi.org/10.3390/ijms21165829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461568PMC
August 2020

Philanthotoxin-343 attenuates retinal and optic nerve injury, and protects visual function in rats with N-methyl-D-aspartate-induced excitotoxicity.

PLoS One 2020 24;15(7):e0236450. Epub 2020 Jul 24.

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Retinal ganglion cell (RGC) loss and optic neuropathy, both hallmarks of glaucoma, have been shown to involve N-methyl-D-aspartate receptor (NMDAR)-mediated excitotoxicity. This study investigated the neuroprotective effects of Philanthotoxin (PhTX)-343 in NMDA-induced retinal injury to alleviate ensuing visual impairments. Sprague-Dawley rats were divided into three; Group I was intravitreally injected with phosphate buffer saline as the control, Group II was injected with NMDA (160 nM) to induce retinal excitotoxic injury, while Group III was injected with PhTX-343 (160 nM) 24 h prior to excitotoxicity induction with NMDA. Rats were subjected to visual behaviour tests seven days post-treatment and subsequently euthanized. Rat retinas and optic nerves were subjected to H&E and toluidine blue staining, respectively. Histological assessments showed that NMDA exposure resulted in significant loss of retinal cell nuclei and thinning of ganglion cell layer (GCL). PhTX-343 pre-treatment prevented NMDA-induced changes where the RGC layer morphology is similar to the control. The numbers of nuclei in the NMDA group were markedly lower compared to the control (p<0.05). PhTX-343 group had significantly higher numbers of nuclei within 100 μm length and 100 μm2 area of GCL (2.9- and 1.7-fold, respectively) compared to NMDA group (p<0.05). PhTX-343 group also displayed lesser optic nerve fibres degeneration compared to NMDA group which showed vacuolation in all sections. In the visual behaviour test, the NMDA group recorded higher total distance travelled, and lower total immobile time and episodes compared to the control and PhTX-343 groups (p<0.05). Object recognition tests showed that the rats in PhTX-343 group could recognize objects better, whereas the same objects were identified as novel by NMDA rats despite multiple exposures (p<0.05). Visual performances in the PhTX-343 group were all comparable with the control (p>0.05). These findings suggested that PhTX-343 inhibit retinal cell loss, optic nerve damage, and visual impairments in NMDA-induced rats.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0236450PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7380593PMC
September 2020

Missing Selectivity of Targeted 4β-Phorbol Prodrugs Expected to be Potential Chemotherapeutics.

ACS Med Chem Lett 2020 May 23;11(5):671-677. Epub 2019 Dec 23.

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Jagtvej 162, DK-2100 Copenhagen, Denmark.

Targeting cytotoxic 4β-phorbol esters toward cancer tissue was attempted by conjugating a 4β-pborbol derivative with substrates for the proteases prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) expressed in cancer tissue. The hydrophilic peptide moiety was hypothesized to prevent penetration of the prodrugs into cells and prevent interaction with PKC. Cleavage of the peptide in cancer tumors was envisioned to release lipophilic cytotoxins, which subsequently penetrate into cancer cells. The 4β-phorbol esters were prepared from 4β-phorbol isolated from seeds, while the peptides were prepared by solid-phase synthesis. Cellular assays revealed activation of PKC by the prodrugs and efficient killing of both peptidase positive as well as peptidase negative cells. Consequently no selectivity for enzyme expressing cells was found.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236034PMC
May 2020

Facile Preparation of PNA-Peptide Conjugates with a Polar Maleimide-Thioether Linkage.

Methods Mol Biol 2020 ;2105:97-118

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Conjugation of a delivery peptide containing a thiol functionality (e.g., a cysteine residue) with a PNA oligomer displaying a single unprotected aliphatic primary amine (e.g., the N-terminus or a C-terminal lysine residue) can be achieved via a one-pot modification with a bisfunctional maleimide linker also displaying a reactive N-hydroxysuccinimidyl ester group (e.g., Mal-PEG2-OSu). Here, an optimized protocol with respect to ratios between the reactants as well as recommended reaction times is presented. Formation and conversion of the maleimide-PNA intermediate was followed by analytical HPLC as exemplified by its conjugation to (KFF)K-Cys-NH. In addition, the reaction time required for direct conversion of a preformed Mal-(CH)-(C=O)-PNA oligomer in the presence of a slight excess of thiol-modified peptide (with a varying degree of sterical hindrance: HS-(CH)-CONH-(KFF)K-NH, (KFF)K-hCys-NH and (KFF)K-Cys-NH) is provided.
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http://dx.doi.org/10.1007/978-1-0716-0243-0_6DOI Listing
January 2021

Fmoc-Based Assembly of PNA Oligomers: Manual and Microwave-Assisted Automated Synthesis.

Methods Mol Biol 2020 ;2105:1-16

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Exploration of PNA-peptide conjugates as potential antisense antibiotics necessitates a fast and efficient synthesis protocols for amounts that facilitate determination of structure-activity relationships and in vivo studies in animal infection models. Fmoc/Boc-protected PNA monomers are here used for assembly of oligomers by optimized protocols involving either a manual synthesis method at room temperature or automated microwave-assisted coupling of monomers on a peptide synthesizer.
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http://dx.doi.org/10.1007/978-1-0716-0243-0_1DOI Listing
January 2021

Antidiabetic xanthones with α-glucosidase inhibitory activities from an endophytic Penicillium canescens.

Fitoterapia 2020 Apr 20;142:104522. Epub 2020 Feb 20.

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark. Electronic address:

Worldwide, 463 million people are affected by diabetes of which the majority is diagnosed with Type 2 Diabetes (T2D). T2D can ultimately lead to retinopathy, nephropathy, nerve damage, and amputation of the lower extremities. α-Glucosidase, responsible for converting starch to monosaccharides, is a key therapeutic target for the management of T2D. However, due to substantial side effects of currently marketed drugs, there is an urgent need for the discovery of new α-glucosidase inhibitors. In our ongoing efforts to identify novel α-glucosidase inhibitors from Nature, we are investigating the potential of endophytic filamentous fungi as sustainable sources of hits and/or leads for future antihyperglycemic drugs. Here we report one previously unreported xanthone (5) and two known xanthones (7 and 11) as α-glucosidase inhibitors, isolated from an endophytic Penicillium canescens, recovered from fruits of Juniperus polycarpos. The three xanthones 5, 7, and 11 showed inhibitory activities against α-glucosidase with IC values of 38.80 ± 1.01 μM, 32.32 ± 1.01 μM, and 75.20 ± 1.02 μM, respectively. Further pharmacological characterization revealed a mixed-mode inhibition for 5, a competitive inhibition for 7, while 11 acted as a non-competitive inhibitor.
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http://dx.doi.org/10.1016/j.fitote.2020.104522DOI Listing
April 2020

Fluorescent Analogues of Human α-Calcitonin Gene-Related Peptide with Potent Vasodilator Activity.

Int J Mol Sci 2020 Feb 17;21(4). Epub 2020 Feb 17.

Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark.

Human α-calcitonin gene-related peptide (h-α-CGRP) is a highly potent vasodilator peptide that belongs to the family of calcitonin peptides. There are two forms of CGRP receptors in humans and rodents: α-CGRP receptor predominately found in the cardiovascular system and β-CGRP receptor predominating in the gastrointestinal tract. The CGRP receptors are primarily localized to C and Aδ sensory fibers, where they are involved in nociceptive transmission and migraine pathophysiology. These fibers are found both peripherally and centrally, with extensive perivascular location. The CGRP receptors belong to the class B G-protein-coupled receptors, and they are primarily associated to signaling via Gα proteins. The objectives of the present work were: (i) synthesis of three single-labelled fluorescent analogues of h-α-CGRP by 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis, and (ii) testing of their biological activity in isolated human, mouse, and rat arteries by using a small-vessel myograph setup. The three analogues were labelled with 5(6)-carboxyfluorescein via the spacer 6-aminohexanoic acid at the chain of Lys or Lys. Circular dichroism (CD) experiments were performed to obtain information on the secondary structure of these fluorescently labelled peptides. The CD spectra indicated that the folding of all three analogues was similar to that of native α-CGRP. The three fluorescent analogues of α-CGRP were successfully prepared with a purity of >95%. In comparison to α-CGRP, the three analogues exhibited similar efficacy, but different potency in producing a vasodilator effect. The analogue labelled at the N-terminus proved to be the most readily synthesized, but it was found to possess the lowest vasodilator potency. The analogues labelled at Lys or Lys exhibited an acceptable reduction in potency (i.e., 3-5 times and 5-10 times less potent, respectively), and thus they have potential for use in further investigations of receptor internalization and neuronal reuptake.
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http://dx.doi.org/10.3390/ijms21041343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072916PMC
February 2020

Peptide/Peptoid Hybrid Oligomers: The Influence of Hydrophobicity and Relative Side-Chain Length on Antibacterial Activity and Cell Selectivity.

Molecules 2019 Dec 4;24(24). Epub 2019 Dec 4.

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Jagtvej 162, DK-2100 Copenhagen, Denmark.

Previous optimisation studies of peptide/peptoid hybrids typically comprise comparison of structurally related analogues displaying different oligomer length and diverse side chains. The present work concerns a systematically constructed series of 16 closely related 12-mer oligomers with an alternating cationic/hydrophobic design, representing a wide range of hydrophobicity and differences in relative side-chain lengths. The aim was to explore and rationalise the structure-activity relationships within a subclass of oligomers displaying variation of three structural features: (i) cationic side-chain length, (ii) hydrophobic side-chain length, and (iii) type of residue that is of a flexible peptoid nature. Increased side-chain length of cationic residues led to reduced hydrophobicity till the side chains became more extended than the aromatic/hydrophobic side chains, at which point hydrophobicity increased slightly. Evaluation of antibacterial activity revealed that analogues with lowest hydrophobicity exhibited reduced activity against , while oligomers with the shortest cationic side chains were most potent against . Thus, membrane-disruptive interaction with appears to be promoted by a hydrophobic surface of the oligomers (comprised of the aromatic groups shielding the cationic side chains). Peptidomimetics with short cationic side chains exhibit increased hemolytic properties as well as give rise to decreased HepG2 (hepatoblastoma G2 cell line) cell viability. An optimal hydrophobicity window could be defined by a threshold of minimal hydrophobicity conferring activity toward and a threshold for maximal hydrophobicity, beyond which cell selectivity was lost.
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http://dx.doi.org/10.3390/molecules24244429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6943742PMC
December 2019

Mechanistic profiling of the release kinetics of siRNA from lipidoid-polymer hybrid nanoparticles in vitro and in vivo after pulmonary administration.

J Control Release 2019 09 6;310:82-93. Epub 2019 Aug 6.

Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark. Electronic address:

Understanding the release kinetics of siRNA from nanocarriers, their cellular uptake, their in vivo biodistribution and pharmacokinetics is a fundamental prerequisite for efficient optimisation of the design of nanocarriers for siRNA-based therapeutics. Thus, we investigated the influence of composition on the siRNA release from lipid-polymer hybrid nanoparticles (LPNs) consisting of cationic lipidoid 5 (L) and poly(DL-lactic-co-glycolic acid) (PLGA) intended for pulmonary administration. An array of siRNA-loaded LPNs was prepared by systematic variation of: (i) the L content (10-20%, w/w), and (ii) the L:siRNA ratio (10,1-30:1, w/w). For comparative purposes, L-based lipoplexes, L-based stable nucleic acid lipid nanoparticles (SNALPs). and dioleoyltrimethylammoniumpropane (DOTAP)-modified LPNs loaded with siRNA were also prepared. Release studies in buffer and lung surfactant-containing medium showed that siRNA release is dependent on the presence of both surfactant and heparin (a displacing agent) in the release medium, since these interact with the lipid shell structure thereby facilitating decomplexation of L and siRNA, as evident from the retarded siRNA release when the L content and the L:siRNA ratio were increased. This confirms the hypothesis that siRNA loaded in LPNs is predominantly present as complexes with the cationic lipid and primarily is located near the particle surface. Cellular uptake and tolerability studies in the human macrophage cell line THP-1 and the type I-like human alveolar epithelial cell line hAELVi, which together represents a monolayer-based barrier model of lung epithelium, indicated that uptake of LPNs was much higher in THP-1 cells in agreement with their primary clearance role. In vivo biodistributions of formulations loaded with Alexa Fluor® 750-labelled siRNA after pulmonary administration in mice were compared by using quantitative fluorescence imaging tomography. The L-modified LPNs, SNALPs and DOTAP-modified LPNs displayed significantly increased lung retention of siRNA as compared to L-based lipoplexes, which had a biodistribution profile comparable to that of non-loaded siRNA, for which >50% of the siRNA dose permeated the air-blood barrier within 6 h and subsequently was excreted via the kidneys. Hence, the enhanced lung retention upon pulmonary administration of siRNA-loaded LPNs represents a promising characteristic that can be used to control the delivery of the siRNA cargo to lung tissue for local management of disease.
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http://dx.doi.org/10.1016/j.jconrel.2019.08.004DOI Listing
September 2019

Identification of Factors of Importance for Spray Drying of Small Interfering RNA-Loaded Lipidoid-Polymer Hybrid Nanoparticles for Inhalation.

Pharm Res 2019 Aug 2;36(10):142. Epub 2019 Aug 2.

Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100, Copenhagen Ø, Denmark.

Background: With the recent approval of the first small interfering RNA (siRNA) therapeutic formulated as nanoparticles, there is increased incentive for establishing the factors of importance for the design of stable solid dosage forms of such complex nanomedicines.

Methods: The aims of this study were: (i) to identify factors of importance for the design of spray-dried siRNA-loaded lipidoid-poly(DL-lactic-co-glycolic acid) hybrid nanoparticles (LPNs), and (ii) to evaluate their influence on the resulting powders by using a quality-by-design approach. Critical formulation and process parameters were linked to critical quality attributes (CQAs) using design of experiments, and an optimal operating space (OOS) was identified.

Results: A series of CQAs were identified based on the quality target product profile. The loading (ratio of LPNs to the total solid content) and the feedstock concentration were determined as critical parameters, which were optimized systematically. Mannitol was chosen as stabilizing excipient due to the low water content of the resulting powders. The loading negatively affected the colloidal stability of the LPNs, whereas feedstock concentration correlated positively with the powder particle size. The optimal mannitol-based solid formulation, defined from the OOS, displayed a loading of 5% (w/w), mass median aerodynamic diameter of 3.3 ± 0.2 μm, yield of 60.6 ± 6.6%, and a size ratio of 1.15 ± 0.03. Dispersed micro-embedded LPNs had preserved physicochemical characteristics as well as in vitro siRNA release profile and gene silencing, as compared to non-spray-dried LPNs.

Conclusion: The optimal solid dosage forms represent robust formulations suitable for higher scale-up manufacturing.
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http://dx.doi.org/10.1007/s11095-019-2663-yDOI Listing
August 2019

Repurposing Azithromycin and Rifampicin Against Gram-Negative Pathogens by Combination With Peptidomimetics.

Front Cell Infect Microbiol 2019 2;9:236. Epub 2019 Jul 2.

Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark.

Synthetic peptidomimetics may be designed to mimic functions of antimicrobial peptides, including potentiation of antibiotics, yet possessing improved pharmacological properties. Pairwise screening of 42 synthetic peptidomimetics combined with the antibiotics azithromycin and rifampicin in multidrug-resistant (MDR) ST131 and ST258 led to identification of two subclasses of α-peptide/β-peptoid hybrids that display synergy with azithromycin and rifampicin (fractional inhibitory concentration indexes of 0.03-0.38). Further screening of the best three peptidomimetics in combination with a panel of 21 additional antibiotics led to identification of peptidomimetics that potentiated ticarcillin/clavulanate and erythromycin against , and clindamycin against . The study of six peptidomimetics was extended to , confirming synergy with antibiotics for five of them. The most promising compound, H-(Lys-βNPhe)-NH, exerted only a minor effect on the viability of mammalian cells (EC ≥ 124-210 μM), and thus exhibited the highest selectivity toward bacteria. This compound also synergized with rifampicin and azithromycin at sub-micromolar concentrations (0.25-0.5 μM), thereby inducing susceptibility to these antibiotics at clinically relevant concentrations in clinical MDR isolates. This peptidomimetic lead and its analogs constitute promising candidates for efficient repurposing of rifampicin and azithromycin against Gram-negative pathogens.
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http://dx.doi.org/10.3389/fcimb.2019.00236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6615261PMC
February 2020

Philanthotoxin Analogues That Selectively Inhibit Ganglionic Nicotinic Acetylcholine Receptors with Exceptional Potency.

J Med Chem 2019 07 24;62(13):6214-6222. Epub 2019 Jun 24.

School of Life Sciences , University of Nottingham , University Park , Nottingham NG7 2RD , U.K.

Philanthotoxin-433 (PhTX-433) is an active component of the venom from the Egyptian digger wasp, . PhTX-433 nonselectively inhibits several excitatory ligand-gated ion channels, and we recently showed that its synthetic analogue, PhTX-343, exhibits strong selectivity for neuronal over muscle-type nicotinic acetylcholine receptors (nAChRs). Here, we examined the action of 17 analogues of PhTX-343 against ganglionic (α3β4) and brain (α4β2) nAChRs expressed in oocytes by using a two-electrode voltage clamp at -100 mV. IC values for PhTX-343 inhibition of α3β4 and α4β2 receptors were 7.7 and 80 nM, respectively. All the studied analogues had significantly higher potency at α3β4 nAChRs with IC values as low as 0.16 nM and with up to 91-fold selectivity for α3β4 over α4β2 receptors. We conclude that PhTX-343 analogues displaying both a saturated ring and an aromatic moiety in the hydrophobic headgroup of the molecule demonstrate exceptional potency and selectivity for α3β4 nAChRs.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00519DOI Listing
July 2019

Lipidoid-polymer hybrid nanoparticles loaded with TNF siRNA suppress inflammation after intra-articular administration in a murine experimental arthritis model.

Eur J Pharm Biopharm 2019 Sep 11;142:38-48. Epub 2019 Jun 11.

Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark. Electronic address:

Rheumatoid arthritis (RA) is a common autoimmune disease, which is characterized by painful chronic inflammation in the joints, and novel safe and efficacious treatments are urgently needed. RNA interference (RNAi) therapy based on small interfering RNA (siRNA) is a promising approach for silencing specific genes involved in inflammation. However, delivery of siRNA to the target site, i.e. the cytosol of immune cells, is a challenge. Here, we designed lipid-polymer hybrid nanoparticles (LPNs) composed of lipidoid and poly(DL-lactic-co-glycolic acid) loaded with a therapeutic cargo siRNA directed against the proinflammatory cytokine tumor necrosis factor (TNF), which plays a key role in the progression of RA. We compared their efficacy and safety with reference lipidoid-based stable nucleic acid lipid particles (SNALPs) in vitro and in vivo. Cryogenic transmission electron microscopy, atomic force microscopy and small-angle X-ray scattering revealed that the mode of loading of siRNA in lamellar structures differs between the two formulations. Thus, siRNA was tightly packed in LPNs, while LPNs displayed lower adhesion than SNALPs. The LPNs mediated a higher TNF silencing effect in vitro than SNALPs in the RAW 264.7 macrophage cell line activated with lipopolysaccharide. For both types of delivery systems, macropinocytosis was involved in cellular uptake. In addition, clathrin-mediated endocytosis contributed to uptake of SNALPs. LPNs loaded with TNF siRNA mediated sequence-specific suppression of inflammation in a murine experimental arthritis model upon intra-articular administration. Hence, the present study demonstrates that LPN-mediated TNF knockdown constitutes a promising approach for arthritis therapy of TNF-mediated chronic inflammatory conditions.
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http://dx.doi.org/10.1016/j.ejpb.2019.06.009DOI Listing
September 2019

Comparison of two different PEGylation strategies for the liposomal adjuvant CAF09: Towards induction of CTL responses upon subcutaneous vaccine administration.

Eur J Pharm Biopharm 2019 Jul 2;140:29-39. Epub 2019 May 2.

Statens Serum Institut, Department of Infectious Disease Immunology, Artillerivej 5, 2300 Copenhagen S, Denmark. Electronic address:

Using subunit vaccines, e.g., based on peptide or protein antigens, to teach the immune system to kill abnormal host cells via induction of cytotoxic T lymphocytes (CTL) is a promising strategy against intracellular infections and cancer. However, customized adjuvants are required to potentiate antigen-specific cellular immunity. One strong CTL-inducing adjuvant is the liposomal cationic adjuvant formulation (CAF)09, which is composed of dimethyldioctadecylammonium (DDA) bromide, monomycoloyl glycerol (MMG) analogue 1 and polyinosinic:polycytidylic acid [poly(I:C)]. However, this strong CTL induction requires intraperitoneal administration because the vaccine forms a depot at the site of injection (SOI) after subcutaneous (s.c.) or intramuscular (i.m.) injection, and depot formation impedes the crucial vaccine targeting to the cross-presenting dendritic cells (DCs) residing in the lymph nodes (LNs). The purpose of the present study was to investigate the effect of polyethylene glycol (PEG) grafting of CAF09 on the ability of the vaccine to induce antigen-specific CTL responses after s.c. administration. We hypothesized that steric stabilization and charge shielding of CAF09 by PEGylation may reduce depot formation at the SOI and enhance passive drainage to the LNs, eventually improving CTL induction. Hence, the vaccine (antigen/CAF09) was post-grafted with a novel type of anionic PEGylated peptides based on GDGDY repeats, which were end-conjugated with one or two PEG moieties, resulting in mono- and bis-PEG-peptides of different lengths (10, 15 and 20 amino acid residues). For comparison, CAF09 was also grafted by inclusion of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(PEG)-2000 (DSPE-PEG) in the bilayer structure during preparation. Grafting of CAF09 with either type of PEG resulted in charge shielding, evident from a reduced surface charge. Upon s.c. immunization of mice with the model antigen ovalbumin (OVA) adjuvanted with PEGylated CAF09, stronger CTL responses were induced as compared to immunization of mice with unadjuvanted OVA. Biodistribution studies confirmed that grafting of CAF09 with DSPE-PEG improved the passive drainage of the vaccine to LNs, because a higher dose fraction was recovered in DCs present in the draining LNs, as compared to the dose fraction detected for non-PEGylated CAF09. In conclusion, PEGylation of CAF09 may be a useful strategy for the design of an adjuvant, which induces CTL responses after s.c. and i.m. administration. In the present studies, CAF09 grafted with 10 mol% DSPE-PEG is the most promising of the tested adjuvants, but additional studies are required to further elucidate the potential of the strategy.
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http://dx.doi.org/10.1016/j.ejpb.2019.04.020DOI Listing
July 2019

F-substituted amino acids as an alternative to fluorophore labels: monitoring of degradation and cellular uptake of analogues of penetratin by F NMR.

J Biomol NMR 2019 Apr 18;73(3-4):167-182. Epub 2019 Mar 18.

Section for Biotechnology, Department of Chemistry and Bioscience, Aalborg University, Frederik Bajers Vej 7H, 9220, Aalborg, Denmark.

Current methods for assessment of cellular uptake of cell-penetrating peptides (CPPs) often rely on detection of fluorophore-labeled CPPs. However, introduction of the fluorescent probe often confers changed physicochemical properties, so that the fluorophore-CPP conjugate may exhibit cytotoxic effects and membrane damage not exerted by the native CPP. In the present study, introduction of fluorine probes was investigated as an alternative to fluorophore labeling of a CPP, since this only confers minor changes to its overall physicochemical properties. The high sensitivity of F NMR spectroscopy and the absence of background signals from naturally occurring fluorine enabled detection of internalized CPP. Also, degradation of fluorine-labeled peptides during exposure to Caco-2 cells could be followed by using F NMR spectroscopy. In total, five fluorinated analogues of the model CPP penetratin were synthesized by using commercially available fluorinated amino acids as labels, including one analogue also carrying an N-terminal fluorophore. The apparent cellular uptake was considerably higher for the fluorophore-penetratin conjugate indicating that the fluorophore moiety promoted uptake of the peptide. The use of F NMR spectroscopy enabled monitoring of the fate of the CPPs over time by establishing molar balances, and by verifying CPP integrity upon uptake. Thus, the NMR-based method offers several advantages over currently widespread methods relying on fluorescence detection. The present findings provide guidelines for improved labeling strategies for CPPs, thereby expanding the repertoire of analytical techniques available for studying degradation and uptake of CPPs.
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http://dx.doi.org/10.1007/s10858-019-00239-3DOI Listing
April 2019

Antimicrobial Activity of α-Peptide/β-Peptoid Lysine-Based Peptidomimetics Against Colistin-Resistant Isolated From Cystic Fibrosis Patients.

Front Microbiol 2019 20;10:275. Epub 2019 Feb 20.

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

infection is a predominant cause of morbidity and mortality in patients with cystic fibrosis infection and with a compromised immune system. Emergence of bacterial resistance renders existing antibiotics inefficient, and therefore discovery of new antimicrobial agents is highly warranted. In recent years, numerous studies have demonstrated that antimicrobial peptides (AMPs) constitute potent agents against a range of pathogenic bacteria. However, AMPs possess a number of drawbacks such as susceptibility to proteolytic degradation with ensuing low bioavailability. To circumvent these undesired properties of AMPs unnatural amino acids or altered backbones have been incorporated to provide stable peptidomimetics with retained antibacterial activity. Here, we report on antimicrobial α-peptide/β-peptoid lysine-based peptidomimetics that exhibit high potency against clinical drug-resistant strains obtained from cystic fibrosis patients. These clinical strains possess and/or mutations that confer high resistance to colistin, the last-resort antibiotic for treatment of infections caused by . The lead peptidomimetic LBP-2 demonstrated a 12-fold improved anti-pseudomonal activity as compared to colistin sulfate as well as favorable killing kinetics, similar antibiofilm activity, and moderate cytotoxicity.
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http://dx.doi.org/10.3389/fmicb.2019.00275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391360PMC
February 2019

Microwave-assisted solid-phase synthesis of antisense acpP peptide nucleic acid-peptide conjugates active against colistin- and tigecycline-resistant E. coli and K. pneumoniae.

Eur J Med Chem 2019 Apr 10;168:134-145. Epub 2019 Feb 10.

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Jagtvej 162, DK-2100, Denmark. Electronic address:

Recent discovery of potent antibacterial antisense PNA-peptide conjugates encouraged development of a fast and efficient synthesis protocol that facilitates structure-activity studies. The use of an Fmoc/Boc protection scheme for both PNA monomers and amino acid building blocks in combination with microwave-assisted solid-phase synthesis proved to be a convenient procedure for continuous assembly of antisense PNA-peptide conjugates. A validated antisense PNA oligomer (CTCATACTCT; targeting mRNA of the acpP gene) was linked to N-terminally modified drosocin (i.e., RXR-PRPYSPRPTSHPRPIRV; X = aminohexanoic acid) or to a truncated Pip1 peptide (i.e., RXRRXR-IKILFQNRRMKWKK; X = aminohexanoic acid), and determination of the antibacterial effects of the resulting conjugates allowed assessment of the influence of different linkers as well as differences between the L- and D-forms of the peptides. The drosocin-derived compound without a linker moiety exhibited highest antibacterial activity against both wild-type Escherichia coli and Klebsiella pneumoniae (MICs in the range 2-4 μg/mL ∼ 0.3-0.7 μM), while analogues displaying an ethylene glycol (eg1) moiety or a polar maleimide linker also possessed activity toward wild-type K. pneumoniae (MICs of 4-8 μg/mL ∼ 0.6-1.3 μM). Against two colistin-resistant E. coli strains the linker-deficient compound proved most potent (with MICs in the range 2-4 μg/mL ∼ 0.3-0.7 μM). The truncated all-L Pip1 peptide had moderate inherent activity against E. coli, and this was unaltered or reduced upon conjugation to the antisense PNA oligomer. By contrast, this peptide was 8-fold less potent against K. pneumoniae, but in this case some PNA-peptide conjugates exhibited potent antisense activity (MICs of 2-8 μg/mL ∼ 0.3-1.2 μM). Most interestingly, the antibacterial activity of the D-form peptide itself was 2- to 16-fold higher than that of the L-form, even for the colistin- and tigecycline-resistant E. coli strains (MIC of 1-2 μg/mL ∼ 0.25-0.5 μM). Low activity was found for conjugates with a two-mismatch PNA sequence corroborating an antisense mode of action. Conjugates containing a D-form peptide were also significantly less active. In conclusion, we have designed and synthesized antisense PNA-drosocin conjugates with potent antibacterial activity against colistin- and tigecycline-resistant E. coli and K. pneumonia without concomitant hemolytic properties. In addition, a truncated D-form of Pip1 was identified as a peptide exhibiting potent activity against both wild-type and multidrug-resistant E. coli, P. aeruginosa, and A. baumannii (MICs within the range 1-4 μg/mL ∼ 0.25-1 μM) as well as toward wild-type Staphylococcus aureus (MIC of 2-4 μg/mL ∼ 0.5-1.0 μM).
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http://dx.doi.org/10.1016/j.ejmech.2019.02.024DOI Listing
April 2019

Macrophage Phosphoproteome Analysis Reveals MINCLE-dependent and -independent Mycobacterial Cord Factor Signaling.

Mol Cell Proteomics 2019 04 11;18(4):669-685. Epub 2019 Jan 11.

From the ‡Institute of Clinical Microbiology, Immunology and Hygiene, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany;. Electronic address:

Immune sensing of relies on recognition by macrophages. Mycobacterial cord factor, trehalose-6,6'-dimycolate (TDM), is the most abundant cell wall glycolipid and binds to the C-type lectin receptor (CLR) MINCLE. To explore the kinase signaling linking the TDM-MINCLE interaction to gene expression, we employed quantitative phosphoproteome analysis. TDM caused upregulation of 6.7% and suppressed 3.8% of the 14,000 phospho-sites identified on 3727 proteins. MINCLE-dependent phosphorylation was observed for canonical players of CLR signaling ( PLCγ, PKCδ), and was enriched for PKCδ and GSK3 kinase motifs. MINCLE-dependent activation of the PI3K-AKT-GSK3 pathway contributed to inflammatory gene expression and required the PI3K regulatory subunit p85α. Unexpectedly, a substantial fraction of TDM-induced phosphorylation was MINCLE-independent, a finding paralleled by transcriptome data. Bioinformatics analysis of both data sets concurred in the requirement for MINCLE for innate immune response pathways and processes. In contrast, MINCLE-independent phosphorylation and transcriptome responses were linked to cell cycle regulation. Collectively, our global analyses show substantial reprogramming of macrophages by TDM and reveal a dichotomy of MINCLE-dependent and -independent signaling linked to distinct biological responses.
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http://dx.doi.org/10.1074/mcp.RA118.000929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442366PMC
April 2019

Application of a Quality-By-Design Approach to Optimise Lipid-Polymer Hybrid Nanoparticles Loaded with a Splice-Correction Antisense Oligonucleotide: Maximising Loading and Intracellular Delivery.

Pharm Res 2019 Jan 9;36(3):37. Epub 2019 Jan 9.

Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2,, DK-2100, Copenhagen Ø, Denmark.

Background: Antisense oligonucleotides (ASOs) are promising therapeutics for specific modulation of cellular RNA function. However, ASO efficacy is compromised by inefficient intracellular delivery. Lipid-polymer hybrid nanoparticles (LPNs) are attractive mediators of intracellular ASO delivery due to favorable colloidal stability and sustained release properties.

Methods: LPNs composed of cationic lipidoid 5 (L) and poly(DL-lactic-co-glycolic acid) were studied for delivery of an ASO mediating splice correction of a luciferase gene transcript (Luc-ASO). Specific purposes were: (i) to increase the mechanistic understanding of factors determining the loading of ASO in LPNs, and (ii) to optimise the LPNs and customise them for Luc-ASO delivery in HeLa pLuc/705 cells containing an aberrant luciferase gene by using a quality-by-design approach. Critical formulation variables were linked to critical quality attributes (CQAs) using risk assessment and design of experiments, followed by delineation of an optimal operating space (OOS).

Results: A series of CQAs were identified based on the quality target product profile. The L content and L:Luc-ASO ratio (w/w) were determined as critical formulation variables, which were optimised systematically. The optimised Luc-ASO-loaded LPNs, defined from the OOS, displayed high loading and mediated splice correction at well-tolerated, lower doses as compared to those required for reference L-based lipoplexes, L-modified stable nucleic acid lipid nanoparticles or LPNs modified with dioleoyltrimethylammonium propane (conventional cationic lipid).

Conclusions: The optimal Luc-ASO-loaded LPNs represent a robust formulation that mediates efficient intracellular delivery of Luc-ASO. This opens new avenues for further development of LPNs as a broadly applicable technology platform for delivering nucleic acid cargos intracellularly.
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http://dx.doi.org/10.1007/s11095-018-2566-3DOI Listing
January 2019

Repurposing azithromycin and rifampicin against Gram-negative pathogens by combination with peptide potentiators.

Int J Antimicrob Agents 2019 Jun 14;53(6):868-872. Epub 2018 Nov 14.

Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark; Department of Biomedical Sciences, Ross University School of Veterinary Medicine, Basseterre, St Kitts.

Gram-negative bacterial pathogens are intrinsically resistant to several antibiotics that are not able to penetrate the cell envelope barrier. The aim of this study was to identify peptides that at low concentrations induce susceptibility to these antibiotics in multidrug-resistant (MDR) Gram-negative bacterial strains of clinical relevance. Pairwise screening of 34 diverse peptides and four antibiotics (erythromycin, linezolid, rifampicin and vancomycin) with primary activity against Gram-positive bacteria identified 4 peptides that at submicromolar concentrations conferred susceptibility to rifampicin or erythromycin in Escherichia coli ATCC 25922. The identified peptides exhibited synergy with azithromycin and potentiated clindamycin in MDR E. coli ST131 and Klebsiella pneumoniae ST258. The low cytotoxicity toward eukaryotic cells (IC > 50 µM) observed for two of these peptides (KLWKKWKKWLK-NH and GKWKKILGKLIR-NH) prompted synthesis and evaluation of the corresponding all-d analogues (D1 and D2), which retained similar synergistic antibacterial profiles. Low concentrations of D1 and D2 in combination with azithromycin and rifampicin inhibited growth of most clinical E. coli, K. pneumoniae and Acinetobacter baumannii strains tested. These data demonstrate that combinatorial screening at low peptide concentrations constitutes an efficient approach to identify clinically relevant peptide-antibiotic combinations. In vivo pharmacokinetic/pharmacodynamic and toxicity studies are needed to further validate the use of the peptides identified in this study for repurposing azithromycin and rifampicin against Gram-negative pathogens.
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http://dx.doi.org/10.1016/j.ijantimicag.2018.10.025DOI Listing
June 2019

Studies on acid stability and solid-phase block synthesis of peptide-peptoid hybrids: ligands for formyl peptide receptors.

Amino Acids 2019 Feb 28;51(2):205-218. Epub 2018 Sep 28.

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Jagtvej 162, 2100, Copenhagen, Denmark.

α-Peptoids as well as peptide/α-peptoid hybrids and peptide/β-peptoid hybrids constitute major classes of proteolytically stable peptidomimetics that have been extensively investigated as mimetics of biologically active peptides. Representatives of lipidated peptide/β-peptoid hybrids have been identified as promising immunomodulatory lead compounds, and hence access to these via protocols suitable for gram-scale synthesis is warranted to enable animal in vivo studies. Recent observations indicated that several byproducts appear in crude mixtures of relatively short benzyl-based peptide/β-peptoid oligomers, and that these were most predominant when the β-peptoid units displayed an α-chiral benzyl side chain. This prompted an investigation of their stability under acidic conditions. Simultaneous deprotection and cleavage of peptidomimetics containing either α-chiral α- or β-peptoid residues required treatment with strong acid only for a short time to minimize the formation of partially debenzylated byproducts. The initial work on peptide/β-peptoid oligomers with an alternating design established that it was beneficial to form the amide bond between the carboxyl group of the α-amino acid and the congested amino functionality of the β-peptoid residue in solution. To further simplify oligomer assembly on solid phase, we now present a protocol for purification-free solid-phase synthesis of tetrameric building blocks. Next, syntheses of peptidomimetic ligands via manual solid-phase methodologies involving tetrameric building blocks were found to give more readily purified products as compared to those obtained with dimeric building blocks. Moreover, the tetrameric building blocks could be utilized in automated synthesis with microwave-assisted heating, albeit the purity of the crude products was not increased.
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http://dx.doi.org/10.1007/s00726-018-2656-xDOI Listing
February 2019

Phorbol Rearrangements.

J Nat Prod 2018 09 14;81(9):2134-2137. Epub 2018 Sep 14.

Department of Drug Design and Pharmacology , University of Copenhagen , Jagtvej 162 , DK-2100 Copenhagen Ø , Denmark.

An alternative procedure for isolation of 4β-phorbol from seeds of Croton tiglium has been developed, and an artifact containing a furan ring formed by rearrangement of 12,13,20- O-triacylated phorbol derivatives into (6b S,7 R,8 R,8a S)-2-(hydroxymethyl)-5,7,9,9-tetramethyl-3,7,8,9,9a,9b-hexahydrocyclopropa[3',4']benzo[1',2':3,4]cyclohepta[1,2- b]furan-6b,8,8a-triol (8a) has been characterized. A mechanism involving an oxidative rearrangement and a decarboxylation for formation of the artifact is proposed.
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http://dx.doi.org/10.1021/acs.jnatprod.8b00607DOI Listing
September 2018

Identification and characterization of a new antifungal peptide in fermented milk product containing bioprotective Lactobacillus cultures.

FEMS Yeast Res 2018 12;18(8)

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark.

Mold and yeast contamination constitutes a major problem in food commodities, including dairy products, hence new natural preventive measures are in high demand. The aim of the current study is to identify and characterize novel antifungal peptides produced by lactic acid bacteria (LAB) in sour cream. By the use of a newly developed image-based 96-well plate fungal growth inhibition assay targeting Debaryomyces hansenii, combined with a range of analytical tools comprising HPLC-high-resolution mass spectrometry, ultrahigh-performance liquid chromatography-Triple Quadrupole MS and nuclear magnetic resonance spectroscopy, we successfully identified a new antifungal peptide (DMPIQAFLLY; 1211 Da) in sour cream enriched with two bioprotective LAB strains. This peptide represents a fragment of casein, the most abundant protein in milk. Presumably, the proteolytic activity of these bioprotective strains results in the observed 4-fold higher concentration of the peptide during storage. Both bioprotective strains are able to generate this peptide in concentrations up to 0.4 μM, independently of the sour cream starter culture employed. The peptide attenuates the growth rate of D. hansenii at concentrations ≥35 μM, and results in smaller cells and more compact colonies. Hence, the peptide is likely contributing to the overall preserving effect of the investigated bioprotective LAB strains.
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http://dx.doi.org/10.1093/femsyr/foy094DOI Listing
December 2018

High-Resolution PTP1B Inhibition Profiling Combined with HPLC-HRMS-SPE-NMR for Identification of PTP1B Inhibitors from .

Molecules 2018 Jul 17;23(7). Epub 2018 Jul 17.

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.

Protein tyrosine phosphatase 1B (PTP1B) is an intracellular enzyme responsible for deactivation of the insulin receptor, and consequently acts as a negative regulator of insulin signal transduction. In recent years, PTP1B has become an important target for controlling insulin resistance and type 2 diabetes. In the present study, the ethyl acetate extract of leaves of (IC = 4.92 µg/mL) was assessed by high-resolution PTP1B inhibition profiling combined with HPLC-HRMS-SPE-NMR for identification of antidiabetic compounds. This disclosed eleven PTP1B inhibitors, including five polyphenolics: 1--()-caffeoyl-4,6-di--galloyl-β-d-glucopyranose (), myricetin 3--α-l-rhamnopyranoside (), quercetin 3--(2″-galloyl)-α-l-rhamnopyranoside (), mearnsetin 3--α-l-rhamnopyranoside (), and kaempferol 3--α-l-arabinopyranoside () as well as eight triterpenoids: maslinic acid (), 3--sumaresinolic acid (), sumaresinolic acid (), 3----coumaroyl maslinic acid (), 3----coumaroyl maslinic acid (), 3----coumaroyl 2α-hydroxydulcioic acid (), oleanolic acid (), and ursolic acid (). These results support the use of as a traditional medicine with antidiabetic properties and its potential as a source of PTP1B inhibitors.
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http://dx.doi.org/10.3390/molecules23071755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6100288PMC
July 2018

Immune responses induced by nano-self-assembled lipid adjuvants based on a monomycoloyl glycerol analogue after vaccination with the Chlamydia trachomatis major outer membrane protein.

J Control Release 2018 09 28;285:12-22. Epub 2018 Jun 28.

Department of Pharmacy, Pharmaceutical Technology and Biopharmacy, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, DE-81377 Munich, Germany. Electronic address:

Nanocarriers based on inverse hexagonal liquid crystalline phases (hexosomes) show promising potential as vaccine delivery systems. Their unique internal structure, composed of both lipophilic domains and water-containing channels, renders them capable of accommodating immunopotentiating compounds and antigens. However, their adjuvant properties are poorly understood. We hypothesized that the supramolecular structure of the lyotropic liquid crystalline phase influences the immunostimulatory activity of lipid-based nanocarriers. To test this, hexosomes were designed containing the lipid phytantriol (Phy) and the immunopotentiator monomycoloyl glycerol-1 (MMG-1). Self-assembly of Phy and MMG-1 into nanocarriers featuring an internal hexagonal phase was confirmed by small-angle X-ray scattering and cryogenic transmission electron microscopy. The effect of the nanostructure on the adjuvant activity was studied by comparing the immunogenicity of Phy/MMG-1 hexosomes with MMG-1-containing lamellar liquid crystalline nanoparticles (liposomes, CAF04). The quality and magnitude of the elicited immune responses were determined after vaccination of CB6/F1 mice using the Chlamydia trachomatis major outer membrane protein (MOMP) as antigen. MMG-1-based hexosomes potentiated significantly stronger MOMP-specific humoral responses than CAF04 liposomes. The liposome-based vaccine formulation induced a much stronger MOMP-specific cell-mediated immune response compared to hexosome-adjuvanted MOMP, which elicited minimal MOMP-specific T-cell stimulation after vaccination. Hence, our data demonstrates that hexosomal and liposomal adjuvants activate the immune system via different mechanisms. Our work provides valuable insights into the adjuvant potential of hexosomes and emphasizes that engineering of the supramolecular structure can be used to design adjuvants with customized immunological properties.
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http://dx.doi.org/10.1016/j.jconrel.2018.06.028DOI Listing
September 2018