TY - JOUR AB - Despite the multifaceted role of leptin for energy homeostasis and its broad therapeutic potential, the FDA/EMA-approved metreleptin constitutes the only leptin drug to date. To translate the promising results from previous studies on murine PASylated leptin with improved solubility and extended plasma half-life using PASylation technology─a biological alternative to PEGylation─we have developed a second-generation human leptin drug candidate and tested it rigorously in vitro and in vivo. To this end, the exposed hydrophobic Trp residue at position 100 in human leptin was replaced by Gln, which, together with the genetic fusion with a 600-residue PAS polypeptide, yielded a protein with high solubility, folding stability and receptor-stimulatory activity. In a pharmacokinetic (PK) study with wild-type mice, this modified human leptin showed an extended plasma half-life of 18.8 ± 3.6 h after subcutaneous (s.c.) injection. Furthermore, leptin-deficient mice were dosed s.c. with the modified human leptin carrying two different PAS fusion tags, PAS#1 or P/A#1, each comprising 600 residues. After only four doses, the disease phenotype, including morbid adiposity, hyperphagia, and hepatic steatosis, was completely reversed by both PASylated leptin versions, but not by the non-PASylated leptin if administered at the same dose. To assess its tissue distribution, P/A(200)-huLeptinW100Q was doubly labeled with two fluorescent dyes, which were specifically attached to the leptin and the PAS moiety, respectively. Analysis of relevant mouse organs by light sheet fluorescence microscopy after clearance revealed colocalized signals in the kidney and liver, thus indicating general stability of the PAS-leptin fusion protein in vivo. However, discrete signals were observed in the hypothalamic region, only with leptin detectable in the choroid plexus, which implies cleavage of the PAS tag during transcytosis across the physiological barriers. This study should pave the way toward a second-generation leptin drug enabling prolonged dosing intervals. AU - Morath, V.* AU - Maurer, S.* AU - Feuchtinger, A. AU - Walser, R.* AU - Schlapschy, M.* AU - Bolze, F.* AU - Metzler, T.* AU - Bruder, J.* AU - Steiger, K.* AU - Walch, A.K. AU - Klingenspor, M.* AU - Skerra, A.* C1 - 74334 C2 - 57479 CY - 1155 16th St, Nw, Washington, Dc 20036 Usa SP - 3017-3032 TI - Long-acting human PASylated leptin reaches the murine central nervous system and offers potential for optimized replacement therapy. JO - Mol. Pharm. VL - 22 IS - 6 PB - Amer Chemical Soc PY - 2025 SN - 1543-8384 ER - TY - JOUR AB - Look-alike and sound-alike (LASA) medications are a critical source of medication errors, posing significant risks to patient safety. This issue is particularly relevant for non-biological complex drugs (NBCDs), whose intricate compositions and manufacturing-dependent properties complicate regulatory assessment and comparability of follow-on products. In this study, we employ high-resolution mass spectrometry (HRMS) techniques, including Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and comprehensive two-dimensional gas chromatography coupled to high-resolution time-of-flight mass spectrometry (GC×GC-HRToF MS), to differentiate between Ichthammol formulations compliant with different pharmacopeia definitions. Despite similar bulk properties, comprehensive molecular-level characterization reveals substantial compositional differences: European Pharmacopeia (Ph. Eur.) compliant samples, which also largely correspond to the U.S. pharmacopeia (USP), predominantly contain sulfonated thiaarenes derived from shale oil, while samples compliant with the Chinese Pharmacopeia (ChP) consist mainly of sulfurized fatty acids and sulfur-linked fatty acid oligomers derived from vegetable oils. Proposed reaction mechanisms describe a classical aromatic sulfonation by sulfuric acid yielding ammonium sulfonates of thiaarenes and, in smaller quantities, ammonium sulfonate arenes in the Ph. Eur. formulations after neutralization with ammonia. In contrast, ChP formulations likely undergo an initial sulfurization via vulcanization, leading to thiophene-containing fatty acids and sulfur-linked oligomers, followed by sulfonation and neutralization. Our findings provide strong evidence for distinct chemical fingerprints, allowing robust differentiation between these complex LASA drugs. These findings were in accordance with the mechanistic pathways for their respective manufacturing processes proposed in this study. These insights highlight the necessity of molecular-level analysis for regulatory assessment of complex pharmaceuticals and underscore the potential risks of relying solely on bulk parameter equivalence in complex drug approval and substitution. AU - Tiemann, O.* AU - Schwalb, L. AU - Hansen, H.J.* AU - Aguilera, M.L.* AU - Gröger, T.M. AU - Huy, P.* AU - Michalik, D.* AU - Gayko, G.* AU - Rüger, C.P.* AU - Zimmermann, R. C1 - 75925 C2 - 58193 CY - 1155 16th St, Nw, Washington, Dc 20036 Usa TI - Regulatory challenges for look-alike and sound-alike non-biological complex drugs: Molecular fingerprint discrimination of Ichthammol formulations. JO - Mol. Pharm. PB - Amer Chemical Soc PY - 2025 SN - 1543-8384 ER - TY - JOUR AB - The endothelin (ET) axis plays a pivotal role in cardiovascular diseases. Enhanced levels of circulating ET-1 have been correlated with an inferior clinical outcome after myocardial infarction (MI) in humans. Thus, the evaluation of endothelin-A receptor (ETAR) expression over time in the course of myocardial injury and healing may offer valuable information toward the understanding of the ET axis involvement in MI. We developed an approach to track the expression of ETAR with a customized molecular imaging probe in a murine model of MI. The small molecular probe based on the ETAR-selective antagonist 3-(1,3-benzodioxol-5-yl)-5-hydroxy-5-(4-methoxyphenyl)-4-[(3,4,5-trimethoxyphenyOmethyl]-2(5H)-furanone (PD156707) was labeled with fluorescent dye, IRDye800cw. Mice undergoing permanent ligation of the left anterior descending artery (LAD) were investigated at day 1, 7, and 21 post surgery after receiving an intravenous injection of the ETAR probe. Cryosections of explanted hearts were analyzed by cryotome-based CCD, and fluorescence reflectance imaging (FRI) and fluorescence signal intensities (SI) were extracted. Fluorescence-mediated tomography (FMT) imaging was performed to visualize probe distribution in the target region in vivo. An enhanced fluorescence signal intensity in the infarct area was detected in cryoCCD images as early as day 1 after surgery and intensified up to 21 days post MI. FRI was capable of detecting significantly enhanced SI in infarcted regions of hearts 7 days after surgery. In vivo imaging by FMT localized enhanced SI in the apex region of infarcted mouse hearts. We verified the localization of the probe and ETAR within the infarct area by immunohistochemistry (IHC). In addition, neovascularized areas were found in the affected myocardium by CD31 staining. Our study demonstrates that the applied fluorescent probe is capable of delineating ETAR expression over time in affected murine myocardium after MI in vivo and ex vivo. AU - Kimm, M.A.* AU - Haas, H.* AU - Stölting, M.* AU - Kuhlmann, M.* AU - Geyer, C.* AU - Glasl, S. AU - Schäfers, M.* AU - Ntziachristos, V. AU - Wildgruber, M.* AU - Höltke, C.* C1 - 57747 C2 - 47902 CY - 1155 16th St, Nw, Washington, Dc 20036 Usa SP - 109-117 TI - Targeting endothelin receptors in a murine model of myocardial infarction using a small molecular fluorescent probe. JO - Mol. Pharm. VL - 17 IS - 1 PB - Amer Chemical Soc PY - 2020 SN - 1543-8384 ER - TY - JOUR AB - Glucagon-like peptide-1 (GLP-1) is an incretin (a type of metabolic hormone that stimulates a decrease in blood glucose levels), holding great potential for the treatment of type 2 diabetes mellitus (T2DM). However, its extremely short half-life of 1-2 min hampers any direct clinical application. Here, we describe the application of the heavy chain of human ferritin (HFt) nanocage as a carrier to improve the pharmacological properties of GLP-1. The GLP-HFt was designed by genetic fusion of GLP-1 to the N-terminus of HFt and was expressed in inclusion bodies in E. coli. The refolding process was developed to obtain a soluble GLP-HFt protein. The biophysical properties determined by size-exclusion chromatography (SEC), dynamic light scattering (DLS), circular dichroism (CD), transmission electron microscopy (TEM), and X-ray crystallography verified that the GLP-HFt successfully formed a 24-mer nanocage with GLP-1 displayed on the external surface of HFt. The in vivo pharmacodynamic results demonstrated that the GLP-HFt nanocage retained the bioactivity of natural GLP-1, significantly reduced the blood glucose levels for at least 24 h in a dose-dependent manner, and inhibited food intake for at least 8-10 h. The half-life of the GLP-HFt nanocage was approximately 52 h in mice after subcutaneous injection. The prolonged half-life and sustained control of blood glucose levels indicate that the GLP-HFt nanocage can be further developed for the treatment of T2DM. Meanwhile, the HFt nanocage proves its great potential as a universal carrier that improves the pharmacodynamic and pharmacokinetic properties of a wide range of therapeutic peptides and proteins. AU - Su, W.* AU - Tan, H.* AU - Janowski, R. AU - Zhang, W.* AU - Wang, P.* AU - Zhang, J.* AU - Zhai, H.* AU - Li, J.* AU - Niessing, D. AU - Sattler, M. AU - Zou, P. C1 - 58843 C2 - 48341 CY - 1155 16th St, Nw, Washington, Dc 20036 Usa SP - 1663-1673 TI - Ferritin-displayed GLP-1 with improved pharmacological activities and pharmacokinetics. JO - Mol. Pharm. VL - 17 IS - 5 PB - Amer Chemical Soc PY - 2020 SN - 1543-8384 ER - TY - JOUR AB - Central nervous system control of metabolic function relies on the input of endocrine messengers from the periphery, including the pancreatic hormone insulin and the adipokine leptin. This concept primarily derives from experiments in animals where substances can be directly applied to the brain. A feasible approach to study the impact of peptidergic messengers on brain function in humans is the intranasal (IN) route of administration, which bypasses the blood-brain barrier and delivers neuropeptides to the brain compartment, but induces considerably less, if any, peripheral uptake than other administration modes. Experimental IN insulin administration has been extensively used to delineate the role of brain insulin signaling in the control of energy homeostasis, but also cognitive function in healthy humans. Clinical pilot studies have found beneficial effects of IN insulin in patients with memory deficits, suggesting that the IN delivery of this and other peptides bears some promise for new, selectively brain-targeted pharmaceutical approaches in the treatment of metabolic and cognitive disorders. More recently, experiments relying on the IN delivery of the hypothalamic hormone oxytocin, which is primarily known for its involvement in psychosocial processes, have provided evidence that oxytocin influences metabolic control in humans. The IN administration of leptin has been successfully tested in animal models but remains to be investigated in the human setting. We briefly summarize the literature on the IN administration of insulin, leptin, and oxytocin, with a particular focus on metabolic effects, and address limitations and perspectives of IN neuropeptide administration. AU - Spetter, M.S.* AU - Hallschmid, M. C1 - 46707 C2 - 37747 CY - Washington SP - 2767-2780 TI - Intranasal neuropeptide administration to target the human brain in health and disease. JO - Mol. Pharm. VL - 12 IS - 8 PB - Amer Chemical Soc PY - 2015 SN - 1543-8384 ER - TY - JOUR AB - Treatment of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease, is hampered by its complex etiology and lack of efficient means for targeted transfer of therapeutics into motoneurons. The objective of this research was engineering of a versatile motoneuron targeting adapter-a full-length atoxic tetanus toxin fused to core-streptavidin (CS-TeTIM)-for retro-axonal transduction of viral vectors; validation of the targeting efficiency of CS-TeTIM in vivo, by expression of green fluorescence protein (GFP) reporter in motoneurons of presymptomatic and symptomatic ALS-like SOD1(G93A) mice, and comparison with age-matched controls; and appraisal of lentiviral transduction with CS-TeTIM relative to (1) a HC binding fragment of tetanus toxin CS-TeTx(HC), (2) rabies glycoprotein (RG), and (3) a CS-TeTIM-RG dual targeting approach. CS-TeTIM and CS-TeTx(HC) were engineered using recombinant technology and site-directed mutagenesis. Biotinylated vectors, pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) or RG, were linked to these adaptors and injected intraperitoneally (ip) into presymptomatic (12 weeks old), symptomatic SOD1(G93A) (22 weeks old) or wild type control mice, followed by monitoring of GFP expression in the spinal cord and supraspinal motor structures with quantitative PCR and immuno-histochemistry. Transcripts were detected in the spinal cord and supraspinal motor structures of all mice 2 weeks after receiving a single ip injection, although in symptomatic SOD1(G93A) animals reporter RNA levels were lower compared to presymptomatic and wild-type controls irrespective of the targeting approach. GFP transduction with CS-TeTIM proved more efficient than CS-TeTx(HC) across all groups while CS-TeTIM-RG dual-targeted vectors yielded the highest transcript numbers. Importantly, in both wild-type and presymptomatic SOD1(G93A) mice strong colabeling of choline-acetyltransferase (ChAT) and GFP was visualized in neurons of the brain stem and spinal cord. CS-TeTIM, a versatile adaptor protein for targeted lentiviral transduction of motoneurons, has been engineered and its competence assessed relative to CS-TeTx(HC) and RG. Evidence has been provided that highlights the potential usefulness of this novel recombinant tool for basic research with implications for improved transfer of therapeutic candidates into motoneurons for the amelioration of ALS and related diseases. AU - O'Leary, V.B. AU - Ovsepian, S.V.* AU - Bodeker, M.* AU - Dolly, J.O.* C1 - 28005 C2 - 32898 SP - 4195-4206 TI - Improved lentiviral transduction of ALS motoneurons in vivo via dual targeting. JO - Mol. Pharm. VL - 10 IS - 11 PB - Amer. Chemical Soc. PY - 2013 SN - 1543-8384 ER - TY - JOUR AB - Genotoxicity information on polymers used for gene delivery is scant, but of great concern, especially when developing polymeric nanocarriers as nonviral vector systems for cancer treatment. The genotoxicity of some engineered nanomaterials, e.g., metal oxides like ZnO, TiO(2), and CuO but also carbon based materials like carbon black or nanotubes, has commonly been related to oxidative stress, and subsequent inflammation. Recent studies of poly(ethylene imine) (PEI)-based polymers, important nonviral vector systems for pDNA and siRNA, might raise concerns because of their toxic effects dominated by cellular oxidative stress and inflammatory responses, similar to the mentioned effects of engineered nanoparticles. In this study, we employed a FE1-MutaMouse lung epithelial cell line based mutation assay to determine the genotoxicity of three PEI-based polymers and nanosized zinc oxide particles (NZO), all of which have previously been shown to trigger oxidative stress and inflammation. In addition, oxidative DNA damage (8-OH-dG) in FE1 cells was assessed by ELISA. The well-known carcinogen benzo[a]pyrene (B[a]P) was used as positive control. FE1 lung epithelial cells were exposed for eight sequential 72 h incubations, and reporter-gene mutation frequency or 8-OH-dG formation was determined to assess mutagenicity and oxidative DNA damage, respectively. No cytotoxic effects were detected at the exposure levels examined, which are representative of PEI concentrations normally used in in vitro transfection studies. In contrast to B[a]P, neither PEI-polymers nor NZO showed any significant mutagenic activity or oxidative DNA damage in the exposed cells, although PEI-based polymers have been shown to generate significant levels of cellular stress and inflammatory responses. We suggest that the lack of any detectable mutagenic/genotoxic activity of the PEI-based polymers studied here is a crucial step toward a safe use of such nanocarriers in clinical trials. AU - Beyerle, A. AU - Long, A.S.* AU - White, P.A.* AU - Kissel, T.* AU - Stöger, T. C1 - 6023 C2 - 28576 SP - 976-981 TI - Poly(ethylene imine) nanocarriers do not induce mutations nor oxidative DNA damage in vitro in mutaMouse FE1 cells. JO - Mol. Pharm. VL - 8 IS - 3 PB - American Chemical Society PY - 2011 SN - 1543-8384 ER - TY - JOUR AB - Polyethylene imine (PEI) based polycations, successfully used for gene therapy or RNA interference in vitro as well as in vivo, have been shown to cause well-known adverse side effects, especially high cytotoxicity. Therefore, various modifications have been developed to improve safety and efficiency of these nonviral vector systems, but profound knowledge about the underlying mechanisms responsible for the high cytotoxicity of PEI is still missing. In this in vitro study, we focused on stress and toxicity pathways triggered by PEI-based vector systems to be used for pulmonary application and two well-known lung toxic particles: fine crystalline silica (CS) and nanosized ZnO (NZO). The cytotoxicity profiles of all stressors were investigated in alveolar epithelial-like type II cells (LA4) to define concentrations with matching toxicity levels (cell viability >60% and LDH release <10%) for subsequent qRT-PCR-based gene array analysis. Within the first 6 h pathway analysis revealed for CS an extrinsic apoptotic signaling (TNF pathway) in contrast to the intrinsic apoptotic pathway (mitochondrial signaling) which was induced by PEI 25 kDa after 24 h treatment. The following causative chain of events seems conceivable: reactive oxygen species derived from particle surface toxicity triggers TNF signaling in the case of CS, whereby endosomal swelling and rupture upon endocytotic PEI 25 kDa uptake causes intracellular stress and mitochondrial alterations, finally leading to apoptotic cell death at higher doses. PEG modification most notably reduced the cytotoxicity of PEI 25 kDa but increased proinflammatory signaling on mRNA and even protein level. Hence in view of the lung as a sensitive target organ this inflammatory stimulation might cause unwanted side effects related to respiratory and cardiovascular disorders. Thus further optimization of the PEI-based vector systems is still needed for pulmonary application. AU - Beyerle, A. AU - Irmler, M. AU - Beckers, J. AU - Kissel, T.* AU - Stöger, T. C1 - 3215 C2 - 27276 SP - 727-737 TI - Toxicity pathway focused gene expression profiling of PEI-based polymers for pulmonary applications. JO - Mol. Pharm. VL - 7 IS - 3 PB - American Chemical Society PY - 2010 SN - 1543-8384 ER - TY - JOUR AB - Oncolytic adenoviruses rank among the most promising innovative agents in cancer therapy. We examined the potential of boosting the efficacy of the oncolytic adenovirus dl520 by associating it with magnetic nanoparticles and magnetic-field-guided infection in multidrug-resistant (MDR) cancer cells in vitro and upon intratumoral injection in vivo. The virus was complexed by self-assembly with core-shell nanoparticles having a magnetite core of about 10 nm and stabilized by a shell containing 68 mass % lithium 3-[2-(perfluoroalkyl)ethylthio]propionate) and 32 mass % 25 kDa branched polyethylenimine. Optimized virus binding, sufficiently stable in 50% fetal calf serum, was found at nanoparticle-to-virus ratios of 5 fg of Fe per physical virus particle (VP) and above. As estimated from magnetophoretic mobility measurements, 3,600 to 4,500 magnetite nanocrystallites were associated per virus particle. Ultrastructural analysis by electron and atomic force microscopy showed structurally intact viruses surrounded by magnetic particles that occasionally bridged several virus particles. Viral uptake into cells at a given virus dose was enhanced 10-fold compared to nonmagnetic virus when infections were carried out under the influence of a magnetic field. Increased virus internalization resulted in a 10-fold enhancement of the oncolytic potency in terms of the dose required for killing 50% of the target cells (IC(50) value) and an enhancement of 4 orders of magnitude in virus progeny formation at equal input virus doses compared to nonmagnetic viruses. Furthermore, the full oncolytic effect developed within two days postinfection compared with six days in a nonmagnetic virus as a reference. Plotting target cell viability versus internalized virus particles for magnetic and nonmagnetic virus showed that the inherent oncolytic productivity of the virus remained unchanged upon association with magnetic nanoparticles. Hence, we conclude that the mechanism of boosting the oncolytic effect by magnetic force is mainly due to the improved internalization of magnetic virus complexes resulting in potentiated virus progeny formation. Upon intratumoral injection and application of a gradient magnetic field in a murine xenograft model, magnetic virus complexes exhibited a stronger oncolytic effect than adenovirus alone. We propose that this approach would be useful during in vivo administration to tumor-feeding blood vessels to boost the efficacy of the primary infection cycle within the tumor. For systemic application, further modification of magnetic adenovirus complexes for shielding and retargeting of the whole magnetic virus complex entity is needed. AU - Tresilwised, N.* AU - Pithayanukul, P.* AU - Mykhaylyk, O.* AU - Holm, P.S.* AU - Holzmüller, R.* AU - Anton, M.* AU - Thalhammer, S. AU - Adigüzel, D. AU - Döblinger, M.* AU - Plank, C.* C1 - 4248 C2 - 27529 SP - 1069-1089 TI - Boosting oncolytic adenovirus potency with magnetic nanoparticles and magnetic force. JO - Mol. Pharm. VL - 7 IS - 4 PB - American Chemical Society PY - 2010 SN - 1543-8384 ER - TY - JOUR AB - This study describes the physicobiological characterization of PEI- and PEG-PEI polyplexes containing partially 2'-OMe modified 25/27mer dicer substrate siRNAs (DsiRNAs) and their in vivo behavior regarding biodistribution and systemic bioavailability after pulmonary application as well as their ability to knock down gene expression in the lung. Biophysical characterization included circular dichroism of siRNA in polyplexes, condensation efficiency of polymers and in vitro stability. After in vivo application, biodistibution and kinetics of radiolabeled polyplexes, were quantified and recorded over time in three-dimensional SPECT images and by end point scintillation counting. The influence on lung tissue and on the humoral and cellular immunosystem was investigated, and finally knockdown of endogenous gene expression in the lung was determined qualitatively. While all of the polymers used in our study were proven to effectively condense siRNA, stability of the complexes depended on the PEG grafting degree. Interestingly, PEI 25 kDa, which showed the least interaction with mucin or surfactant in vitro, performed poorly in vivo. Our nuclear imaging approach enabled us to follow biodistribution of the instilled nanocarriers over time and indicated that PEGylated nanocarriers are more suitable for lung application. While moderate proinflammatory effects were attributed to PE125k-PEG(2k)(10) nanocarriers, none of the treatments caused histological abnormalities. Our preliminary in vivo knockdown experiment suggests that PEG-PEI/siRNA complexes are promising nanomedicines for pulmonary siRNA delivery. These results encouraged us to further investigate possible adverse effects and to quantify in vivo gene silencing in the lung after intratracheal instillation of PEG-PEI/siRNA complexes. AU - Merkel, O.M.* AU - Beyerle, A. AU - Librizzi, D.* AU - Pfestroff, A.* AU - Behr, T.M.* AU - Sproat, B.* AU - Barth, P.J.* AU - Kissel, T.* C1 - 1052 C2 - 26865 SP - 1246-1260 TI - Nonviral siRNA delivery to the lung: Investigation of PEG-PEI polyplexes and their in vivo performance. JO - Mol. Pharm. VL - 6 IS - 4 PB - Amer Chemical Soc PY - 2009 SN - 1543-8384 ER -