High Stability In Plasma Research Articles

Cytotoxic and Antiproliferative Activity of LASSBio-2208 and the Attempts to Determine Its Drug Metabolism and Pharmacokinetics In Vitro Profile.

Inappropriate expression of histone deacetylase (HDAC-6) and deregulation of the phosphatidylinositol 3-kinase (PI3K) signalling pathway are common aberrations observed in cancers. LASSBio-2208, has been previously described as a dual inhibitor in the nanomolar range of HDAC-6 and PI3Kα and is three times more potent in inhibiting HDAC-6. In this paper we described the cytotoxic and antiproliferative potency of LASSBio-2208 on different tumour cell lines, its possible synergism effect in association with PI3K and HDAC-6 inhibitors, and its drug metabolism and pharmacokinetics (DMPK) in vitro profile. Our studies have demonstrated that LASSBio-2208 has moderate cytotoxic potency on breast cancer cell line MCF-7 (IC50 = 23 µM), human leukaemia cell line CCRF-CEM (IC50 = 8.54 µM) and T lymphoblast cell line MOLT-4 (IC50 = 7.15 µM), with no cytotoxic effect on human peripheral blood mononuclear cells (hPBMC). In addition, it has a good antiproliferative effect on MCF-7 cells (IC50 = 5.44 µM), low absorption by parallel artificial membrane permeability-gastrointestinal tract (PAMPA-GIT) and low permeation by parallel artificial membrane permeability-blood-brain barrier (BBB) (PAMPA-BBB), exhibiting high metabolic stability in rat plasma. Moreover, LASSBio-2208 exhibited synergism when combined with getadolisib and tubastatin A, using the concentrations corresponding to their CC50 values on MOLT-4 and CCRF-CEM cells.

SCancerRNA: Expression at the Single-cell Level and Interaction Resource of Non-coding RNA Biomarkers for Cancers.

Non-coding RNAs (ncRNAs) participate in multiple biological processes associated with cancers as tumor suppressors or oncogenic drivers. Due to their high stability in plasma, urine, and many other fluids, ncRNAs have the potential to serve as key biomarkers for early diagnosis and screening of cancers. During cancer progression, tumor heterogeneity plays a crucial role, and it is particularly important to understand the gene expression patterns of individual cells. With the development of single-cell RNA sequencing (scRNA-seq) technologies, uncovering gene expression in different cell types for human cancers has become feasible by profiling transcriptomes at the cellular level. However, a well-organized and comprehensive online resource that provides access to the expression of genes corresponding to ncRNA biomarkers in different cell types at the single-cell level is not available yet. Therefore, we developed the SCancerRNA database to summarize experimentally supported data on long ncRNA, microRNA, PIWI-interacting RNA, small nucleolar RNA, and circular RNA biomarkers, as well as data on their differential expression at the cellular level. Furthermore, we collected biological functions and clinical applications of biomarkers to facilitate the application of ncRNA biomarkers to cancer diagnosis, as well as the monitoring of progression and targeted therapies. SCancerRNA also allows users to explore interaction networks of different types of ncRNAs, and build computational models in the future. SCancerRNA is freely accessible at http://www.scancerrna.com/BioMarker.

Inhibition of N-type calcium channels by phenoxyaniline and sulfonamide analogues.

Building on previous investigations, structural modifications to the neuronal calcium ion channel blocker MONIRO-1 and related compounds were conducted that included replacement of the amide linker with an aniline and isosteric sulfonamide moiety, and the previously used strategy of substitution of the guanidinium group with less hydrophilic amine functionalities. A comprehensive SAR study revealed a number of phenoxyaniline and sulfonamide compounds that were more potent or had similar potency for the CaV2.2 and CaV3.2 channel compared to MONIRO-1 when evaluated in a FLIPR-based intracellular calcium response assay. Cytotoxicity investigations indicated that the sulfonamide analogues were well tolerated by Cos-7 cells at dosages required to inhibit both calcium ion channels. The sulfonamide derivatives were the most promising CaV2.2 inhibitors developed by us to date due, possessing high stability in plasma, low toxicity (estimated therapeutic index > 10), favourable CNS MPO scores (4.0-4.4) and high potency and selectivity, thereby, making this class of compounds suitable candidates for future in vivo studies.

Photosubstitution and photoreduction of a diazido platinum(IV) anticancer complex.

The hyphenation of HPLC with its high separation ability and ICP-MS with its excellent sensitivity, allows the analysis of Pt drugs in biological samples at the low nanomolar concentration levels. On the other hand, LC-MS provides molecular structural confirmation for each species. Using a combination of these methods, we have investigated the speciation of the photoactive anticancer complex diazido Pt(IV) complex trans, trans, trans-[Pt(N3)2(OH)2(py)2] (FM-190) in aqueous solution and biofluids at single-digit nanomolar concentrations before and after irradiation. FM-190 displays high stability in human blood plasma in the dark at 37 °C. Interestingly, the polyhydroxido species [{PtIV(py)2(OH)4} + Na]+ and [{PtIV(py)2(N3)(OH)3} + Na]+ resulting from the replacement of azido ligands, as determined by LC-MS, were the major products after photoirradiation of FM-190 with blue light (463 nm). This finding suggests that such photosubstituted Pt(IV) tri- and tetra-hydroxido species could play important roles in the biological activity of this anticancer complex. Density Functional Theory (DFT) and Time-Dependent DFT (TDDFT) calculations show that these Pt(IV) species arising from FM-190 in aqueous media can be formed directly from a singlet excited state. The results highlight how speciation analysis (metallomics) can shed light on photoactivation pathways for FM-190 and formation of potential excited-state pharmacophores. The ability to detect and identify photoproducts at physiologically-relevant concentrations in cells and tissues will be important for preclinical development studies of this class of photoactivatable platinum drugs.

Harnessing Nanomedicine to Potentiate the Chemo-Immunotherapeutic Effects of Doxorubicin and Alendronate Co-Encapsulated in Pegylated Liposomes.

Encapsulation of Doxorubicin (Dox), a potent cytotoxic agent and immunogenic cell death inducer, in pegylated (Stealth) liposomes, is well known to have major pharmacologic advantages over treatment with free Dox. Reformulation of alendronate (Ald), a potent amino-bisphosphonate, by encapsulation in pegylated liposomes, results in significant immune modulatory effects through interaction with tumor-associated macrophages and activation of a subset of gamma-delta T lymphocytes. We present here recent findings of our research work with a formulation of Dox and Ald co-encapsulated in pegylated liposomes (PLAD) and discuss its pharmacological properties vis-à-vis free Dox and the current clinical formulation of pegylated liposomal Dox. PLAD is a robust formulation with high and reproducible remote loading of Dox and high stability in plasma. Results of biodistribution studies, imaging with radionuclide-labeled liposomes, and therapeutic studies as a single agent and in combination with immune checkpoint inhibitors or gamma-delta T lymphocytes suggest that PLAD is a unique product with distinct tumor microenvironmental interactions and distinct pharmacologic properties when compared with free Dox and the clinical formulation of pegylated liposomal Dox. These results underscore the potential added value of PLAD for chemo-immunotherapy of cancer and the relevance of the co-encapsulation approach in nanomedicine.

Synthesis and evaluation of 111 In-labeled tetrapeptide-based compounds as single-photon emission computed tomography imaging probes targeting granzyme B.

Granzyme B is an attractive target as a biomarker for contributing to improve the treatment with immune checkpoint inhibitor (ICI). In this study, we designed novel 111 In-labeled granzyme B-targeting single-photon emission computed tomography (SPECT) imaging probes, [111 In]IDT and [111 In]IDAT. Nonradioactive In-labeled granzyme B-targeting compounds ([nat In]IDT, [nat In]IDAT) showed the affinity for recombinant mouse granzyme B. [111 In]IDT and [111 In]IDAT were obtained with moderate radiochemical yield and high stability in mouse plasma (>95%). In a biodistribution experiment using tumor-bearing mice, [111 In]IDT and [111 In]IDAT showed moderate accumulation in tumor. Ex vivo autoradiography (ARG) indicated that the accumulation of radioactivity in tumor was correlated to expression of granzyme B confirmed by the immunohistochemical staining. These results indicated that [111 In]IDT and [111 In]IDAT showed the basic properties as granzyme B-targeting SPECT probes.

Quinazoline-2-Carboxamides as Selective PET Radiotracers for Matrix Metalloproteinase-13 Imaging in Atherosclerosis.

Matrix metalloproteinase-13 (MMP-13) plays a critical role in the progression of unstable atherosclerosis. A series of highly potent and selective MMP-13 inhibitors were synthesized around a quinazoline-2-carboxamide scaffold to facilitate radiolabeling with fluorine-18 or carbon-11 positron-emitting nuclides and visualization of atherosclerotic plaques. In vitro enzyme inhibition assays identified three compounds as promising radiotracer candidates. Efficient automated radiosyntheses provided [11C]5b, [11C]5f, and [18F]5j and enabled pharmacokinetic characterization in atherosclerotic mice. The radiotracers displayed substantial differences in their distribution and excretion. Most favorably for vascular imaging, [18F]5j exhibited low uptake in metabolic organs with minimal retention of myocardial radioactivity, substantial renal clearance, and high metabolic stability in plasma. Ex vivo aortic autoradiography and competition studies revealed that [18F]5j specifically binds to MMP-13 within atherosclerotic plaques and localizes to lipid-rich regions. This study demonstrates the utility of the quinazoline-2-carboxamide scaffold for MMP-13 selective positron emission tomography (PET) radiotracer development and identifies [18F]5j for imaging atherosclerosis.

Abstract 2923: JAB-X1800: A potent immunostimulatory antibody-drug conjugate (iADC) targeting CD73

Abstract Background: STING emerges as a key immune sensor and activated STING pathway plays a key role in innate and adaptive immune response. STING agonist has shown promising potency to stimulate anti-tumor immunity. Considering immune-related toxicity due to systemic administration, as well as difficulty in intra-tumor administration, targeted delivery of STING agonist by iADC is warranted. We developed CD73-STING iADC JAB-X1800, which uses anti-CD73 mAb for targeted delivery of payload JAB-27670, a highly potent non-cyclic dinucleotide STING agonist. Method: Binding assay and HEK293-STING-luciferase reporter assay were performed to test affinity of JAB-27670. Tumor cells with CD73 expression and THP-1 cells were co-cultured in the presences of iADC JAB-X1800 to assess the IFN-β production. The cell killing activity of iADC JAB-X1800 was evaluated when cancer cells co-cultured with human PBMCs. Furthermore, human and mouse plasma stability of iADC JAB-X1800 was assessed. Finally, the anti-tumor effects of iADC JAB-X1800 alone or in combination with anti-PD-1 were evaluated in animal models. Results: JAB-27670 is a highly potent STING agonist with sub-nanomolar EC50 in binding and luciferase reporter assays. In CD73-expressing cancer cells and THP-1 co-culture experiments, iADC JAB-X1800 induced IFN-β production with significant improvement compared with the free payload. JAB-X1800 also induced potent killing in vitro. JAB-X1800 is stable in human and mouse plasma. A single dose of JAB-X1800 induced tumor regression in MDA-MB-231 xenograft model and hCD73-MC38 syngeneic model. JAB-X1800 conferred immunologic memory when tumor free mice were re-challenged. JAB-X1800 showed synergistic anti-tumor activity with anti-PD-1 in hCD73-MC38 syngeneic model. Conclusions: The CD73-targeted STING agonist iADC JAB-X1800 demonstrated high stability in plasma, robust activity, and good tolerability in animal models, supporting clinical development of JAB-X1800 alone and in combination with anti-PD-1 for patients with CD73-expressing tumors. Citation Format: Yang Ye, Guiqun Yang, Hongling Wang, Xin Sun, Yanping Wang, Fashun Yan, Lei Wang, Wei Long. JAB-X1800: A potent immunostimulatory antibody-drug conjugate (iADC) targeting CD73 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2923.

Binding Parameters of [11C]MPC-6827, a Microtubule-Imaging PET Radiopharmaceutical in Rodents.

Impairment and/or destabilization of neuronal microtubules (MTs) resulting from hyper-phosphorylation of the tau proteins is implicated in many pathologies, including Alzheimer's disease (AD), Parkinson's disease and other neurological disorders. Increasing scientific evidence indicates that MT-stabilizing agents protect against the deleterious effects of neurodegeneration in treating AD. To quantify these protective benefits, we developed the first brain-penetrant PET radiopharmaceutical, [11C]MPC-6827, for in vivo quantification of MTs in rodent and nonhuman primate models of AD. Mechanistic insights revealed from recently reported studies confirm the radiopharmaceutical's high selectivity for destabilized MTs. To further translate it to clinical settings, its metabolic stability and pharmacokinetic parameters must be determined. Here, we report in vivo plasma and brain metabolism studies establishing the radiopharmaceutical-binding constants of [11C]MPC-6827. Binding constants were extrapolated from autoradiography experiments; pretreatment with a nonradioactive MPC-6827 decreased the brain uptake >70%. It exhibited ideal binding characteristics (typical of a CNS radiopharmaceutical) including LogP (2.9), Kd (15.59 nM), and Bmax (11.86 fmol/mg). Most important, [11C]MPC-6827 showed high serum and metabolic stability (>95%) in rat plasma and brain samples.

Design, Synthesis, and Anti-Bacterial Evaluation of Tetrahydrobenzothiophene Derivatives as Lipopolysaccharide Biogenesis Inhibitors

Background: Bacterial infections caused by multidrug-resistant bacteria have emerged as major threats to human communities worldwide. There is a great need to develop new mechanisms by which anti-bacterial agents can combat bacterial antibiotic resistance. Objective: This study aims to synthesize and characterize a series of novel tetrahydrobenzothiophene derivatives and evaluate their anti-bacterial activities. Method: In this study, we have synthesized 2-benzamido-4,5,6,7-tetrahydrobenzo[b]thiophene-3- carboxylic acid derivatives (3a-3r) and investigated their anti-bacterial activities against E. coli, P. aeruginosa, Salmonella, and S. aureus. Results: The MIC values demonstrated that all synthesized derivatives possessed potent anti-bacterial activity properties. Compounds 3b, 3e, 3f, 3g, 3h, 3n, and 3q exhibited in vitro excellent anti-bacterial efficiency. Compounds 3b, 3e, 3f, and 3p were evaluated by in vitro time-kill assay; they displayed concentration- dependent bacteriostatic effects. Compounds 3b, 3e, 3f, and 3p showed moderate water solubility, high stability in plasma, and moderate acute oral toxicity. Conclusion: Most of the 2-benzamido-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid derivatives exhibited potent anti-bacterial activities. The data reported in this paper may guide the design of tetrahydrobenzothiophene derivatives.

Coumarin derivatives with potential anticancer and antibacterial activity: Design, synthesis, VEGFR-2 and DNA gyrase inhibition, and in silico studies.

A series of coumarin derivatives were designed, synthesized, and evaluated for their antiproliferative activity. Compound 3e exhibited significant antiproliferative activity and was further evaluated at five doses at the National Cancer Institute. It effectively inhibited vascular endothelial growth factor receptor-2 (VEGFR-2) with an IC50 value of 0.082 ± 0.004 µM compared with sorafenib. While compound 3e significantly downregulated total VEGFR-2 and its phosphorylation, it markedly reduced the HUVEC's migratory potential, resulting in a significant disruption in wound healing. Furthermore, compound 3e caused a 22.51-fold increment in total apoptotic level in leukemia cell line HL-60(TB) and a 6.91-fold increase in the caspase-3 level. Compound 3e also caused cell cycle arrest, mostly at the G1/S phase. Antibacterial activity was evaluated against Gram-positive and Gram-negative bacterial strains. Compound 3b was the most active derivative, with the same minimum inhibitory concentration and minimum bactericidal concentration value of 128 μg/mL against K. pneumonia and high stability in mammalian plasma. Moreover, compounds 3b and 3f inhibited Gram-negative DNA gyrase with IC50 = 0.73 ± 0.05 and 1.13 ± 0.07 µM, respectively, compared to novobiocin with an IC50 value of 0.17 ± 0.02 µM. The binding affinity and pattern of derivative 3e toward the VEGFR-2 active site and compounds 3a-c and 3f in the DNA gyrase active site were evaluated using molecular modeling. Overall, ADME studies of the synthesized coumarin derivatives displayed promising pharmacokinetic properties.

Targeting the Gastrin-Releasing Peptide Receptor (GRP-R) in Cancer Therapy: Development of Bombesin-Based Peptide-Drug Conjugates.

Targeted tumour therapy has proved to be an efficient alternative to overcome the limitations of conventional chemotherapy. Among several receptors upregulated in cancer cells, the gastrin-releasing peptide receptor (GRP-R) has recently emerged as a promising target for cancer imaging, diagnosing and treatment due to its overexpression on cancerous tissues such as breast, prostate, pancreatic and small-cell lung cancer. Herein, we report on the in vitro and in vivo selective delivery of the cytotoxic drug daunorubicin to prostate and breast cancer, by targeting GRP-R. Exploiting many bombesin analogues as homing peptides, including a newly developed peptide, we produced eleven daunorubicin-containing peptide-drug conjugates (PDCs), acting as drug delivery systems to safely reach the tumour environment. Two of our bioconjugates revealed remarkable anti-proliferative activity, an efficient uptake by all three tested human breast and prostate cancer cell lines, high stability in plasma and a prompt release of the drug-containing metabolite by lysosomal enzymes. Moreover, they revealed a safe profile and a consistent reduction of the tumour volume in vivo. In conclusion, we highlight the importance of GRP-R binding PDCs in targeted cancer therapy, with the possibility of further tailoring and optimisation.

Abstract A039: Novel tetrazolo-pyridazine based MACC1 transcriptional inhibitors as promising anti-metastatic therapy

Abstract The leading cause of cancer related deaths is the formation of metastasis, frequently caused by insufficient therapies and limited therapy options. Novel compounds able to interfere with metastasis formation are therefore of tremendous interest. Colorectal Cancer (CRC) is the third most prevalent and second most lethal cancer worldwide. In CRC, metastasis formation is linked to poor patient survival and treatment failure. Up to 90% of CRC related deaths are attributed to metastasis formation. Identification of causative drivers of metastasis represents the basis for effective anti-metastatic therapy. Our lab newly identified the gene MACC1 (Metastasis Associated in Colon Cancer 1) in 2009. Since then, MACC1 has been established as a key causal molecule for tumor progression and metastasis formation. It was shown that MACC1 can function as a stage independent prognostic marker, predicting the onset of metastasis in stages I, II and III, based on tumor tissue analyses or through a blood based test with an accuracy of up to 85%. MACC1 promotes several cancer hallmark capabilitites, providing cells with a malignant phenotype. Further, MACC1 has been established as a prognostic and predictive biomarker for metastasis in CRC and more than 20 other solid cancer entities. We therefore searched for novel compounds targeting MACC1 transcription. A high-throughput screen employing HCT116 cells stably transfected with a MACC1 promoter-luciferase reporter construct with more than 118,500 compounds was conducted at the EMBL in Heidelberg. The screen revealed a Tetrazolo-pyridazine based compound as a promising lead for effective inhibition of MACC1 expression. We demonstrated that several SAR and Medchem-generated analogues of our lead compound effectively inhibit MACC1 gene expression and MACC1 driven cancer cell motility in vitro in CRC and cross entity cell lines. Further, they inhibit MACC1-induced tumor progression and metastasis in vivo in a CRC xenograft model in mice. Moreover, ADMET studies were conducted, confirming our compounds are likely to be orally active drugs with high stability in human plasma, low plasma protein binding and great permeability with neglectable efflux in MDR1-MDCKII assay. Through RNA-sequencing and subsequent gene set enrichment analysis a first hypothesis on the mode of action was shaped. An immune pathway has been identified as the most promising signaling pathway targeted by these compounds and is currently explored through knock down and signaling studies. Taken together, this novel class of small molecules represents promising candidates for anti-metastatic therapy in CRC and other solid cancer patients in a personalized medicine setting. Funding: This study is adviced and financed by the SPARK BIH Program Citation Format: Paul Curtis Schöpe, Shixian Yan, Dennis Kobelt, Joe Lewis, Kerstin Putzker, Ulrike Uhrig, Edgar Specker, Jens Peter von Kries, Mathias Dahlmann, Hector E Sanchez-Ibarra, Anke Unger, Mia-Lisa Zischinsky, Bert Klebl, Peter Lindemann, Wolfgang Walther, Marc Nazaré, Ulrike Stein. Novel tetrazolo-pyridazine based MACC1 transcriptional inhibitors as promising anti-metastatic therapy [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A039.

Targeted delivery of alcohol-containing payloads with antibody-drug conjugates.

We herein describe the cell-specific release of alcohol-containing payloads via a sulfatase-sensitive linker in antibody-drug conjugates (ADCs). The linker shows efficient sulfatase-mediated release and high stability in human and mouse plasma. In vitro evaluation demonstrates potent antigen dependent toxicity towards breast cancer cell lines.

Differences in Human Plasma Protein Interactions between Various Polymersomes and Stealth Liposomes as Observed by Fluorescence Correlation Spectroscopy.

A significant factor hindering the clinical translation of polymersomes as vesicular nanocarriers is the limited availability of comparative studies detailing their interaction with blood plasma proteins compared to liposomes. Here, polymersomes are self-assembled via film rehydration, solvent exchange, and polymerization-induced self-assembly using five different block copolymers. The hydrophilic blocks are composed of anti-fouling polymers, poly(ethylene glycol) (PEG) or poly(2-methyl-2-oxazoline) (PMOXA), and all the data is benchmarked to PEGylated "stealth" liposomes. High colloidal stability in human plasma (HP) is confirmed for all but two tested nanovesicles. In situ fluorescence correlation spectroscopy measurements are then performed after incubating unlabeled nanovesicles with fluorescently labeled HP or the specific labeled plasma proteins, human serum albumin, and clusterin (apolipoprotein J). The binding of HP to PMOXA-polymersomes could explain their relatively short circulation times found previously. In contrast, PEGylated liposomes also interact with HP but accumulate high levels of clusterin, providing them with their known prolonged circulation time. The absence of significant protein binding for most PEG-polymersomes indicates mechanistic differences in protein interactions and associated downstream effects, such as cell uptake and circulation time, compared to PEGylated liposomes. These are key observations for bringing polymersomes closer to clinical translation and highlighting the importance of such comparative studies.

Antibacterial and Antifungal Properties of a Novel Antimicrobial Peptide GK-19 and Its Application in Skin and Soft Tissue Infections Induced by MRSA or Candida albicans.

The increasing resistance of human pathogens promotes the development of novel antimicrobial agents. Due to the physical bactericidal mechanism of membrane disruption, antimicrobial peptides are considered as potential therapeutic candidates without inducing microbial resistance. Scorpion venom-derived peptide, Androctonus amoreuxi Antimicrobial Peptide 1 (AamAP1), has been proved to have broad-spectrum antimicrobial properties. However, AamAP1 can induce hemolysis and shows strong toxicity against mammalian cells. Herein, the antimicrobial activity and mechanism of a novel synthetic antimicrobial peptide, GK-19, derived from AamAP1 and its derivatives, was evaluated. Five bacteria and three fungi were used to evaluate the antimicrobial effects of GK-19 in vitro. Scalded mice models combined with skin and soft tissue infections (SSTIs) were used to evaluate its applicability. The results indicated that GK-19 could not only inhibit Gram-positive and Gram-negative bacterial growth, but also kill fungi by disrupting the microbial cell membrane. Meanwhile, GK-19 showed negligible toxicity to mammalian cells, low hemolytic activity and high stability in plasma. Furthermore, in scalded mice models combined with SSTIs induced by either Methicillin-Resistant Staphylococcus aureus (MRSA) or Candida albicans, GK-19 showed significant antimicrobial and healing effects. Overall, it was demonstrated that GK-19 might be a promising drug candidate in the battle against drug-resistant bacterial and fungal infections.

Novel pharmacological effects of lecithinized superoxide dismutase on ischemia/reperfusion injury in the kidneys of mice

Renal ischemia/reperfusion (I/R) injury is a major clinical problem because it can cause acute kidney injury (AKI) or lead to the transition from AKI to chronic kidney disease (CKD). Oxidative stress, which involves the production of reactive oxygen species (ROS), plays an important role in the development and exacerbation of I/R-induced kidney injury. However, we have previously reported that lecithinized superoxide dismutase (PC-SOD), a SOD derivative with high tissue affinity and high stability in plasma, has beneficial effects in various disease models because of its inhibitory effect on ROS production. Therefore, we aimed to determine the effects of intravenous PC-SOD administration in a mouse model of renal injury induced by I/R. PC-SOD markedly ameliorated the I/R-induced increases in markers of renal damage (urea nitrogen, creatinine, neutrophil gelatinase-associated lipocalin, and interleukin-6) and tubular necrosis 48 h after the intervention. We also found that PC-SOD significantly ameliorated the I/R-induced increase in ROS production, using an ex vivo imaging system. Furthermore, PC-SOD inhibited the increases in expression of markers of fibrosis (α-smooth muscle actin and collagen 1A1) 96 h after, and renal fibrosis 25 days after I/R was induced. Finally, we found that PC-SOD ameliorated the I/R-induced AKI in mice with high-fat diet-induced prediabetes. These results suggest that PC-SOD inhibits AKI and the transition from AKI to CKD through the inhibition of ROS production. Therefore, we believe that PC-SOD may represent an effective therapeutic agent for I/R-induced renal injury.

Stability and Safety of Inhibitor Cystine Knot Peptide, GTx1-15, from the Tarantula Spider Grammostola rosea.

Inhibitor cystine knot (ICK) peptides are knotted peptides with three intramolecular disulfide bonds that affect several types of ion channels. Some are proteolytically stable and are promising scaffolds for drug development. GTx1-15 is an ICK peptide that inhibits the voltage-dependent calcium channel Cav3.1 and the voltage-dependent sodium channels Nav1.3 and Nav1.7. As a model molecule to develop an ICK peptide drug, we investigated several important pharmaceutical characteristics of GTx1-15. The stability of GTx1-15 in rat and human blood plasma was examined, and no GTx1-15 degradation was observed in either rat or human blood plasma for 24 h in vitro. GTx1-15 in blood circulation was detected for several hours after intravenous and intramuscular administration, indicating high stability in plasma. The thermal stability of GTx1-15 as examined by high thermal incubation and protein thermal shift assays indicated that GTx1-15 possesses high heat stability. The cytotoxicity and immunogenicity of GTx1-15 were examined using the human monocytic leukemia cell line THP-1. GTx1-15 showed no cytotoxicity or immunogenicity even at high concentrations. These results indicate that GTx1-15 itself is suitable for peptide drug development and as a peptide library scaffold.

Oligo(para-phenylenes)s-Oligoarginine Conjugates as Effective Antibacterial Agents with High Plasma Stability and Low Hemolysis.

Bacterial infections have become a global threat to human health, and the design of antibacterial agents is always an urgent task for biomedicine. Amphiphilic antibacterial agents with a different mechanism of action from traditional antibiotics have attracted researchers' attention more and more in recent years. In this work, a series of antibacterial conjugates composed of oligo(para-phenylenes)s and oligoarginine were synthesized, and their antibacterial activity was investigated. 2,2'-Biphenyl, 2,2″-terphenyl, and 2,2‴-quaterphenyl were conjugated with one or two triarginines by "click" chemical reactions to form compounds. The conjugates showed antibacterial activity against the typical Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria, relatively low cytotoxicity to L929 cell line, and hemolytic activity in a certain range of concentration. Among these conjugates, 2,2‴-quaterphenyl-triarginine conjugate (2,2‴-QP-1) showed the highest antibacterial activity against both E. coli and S. aureus. Besides, it presented better stability in plasma compared with the positive control peixiganan. The antimicrobial mechanism of 2,2‴-QP-1 was also investigated by transmission electron microscopy and confocal laser scanning microscopy, showing that 2,2‴-QP-1 could interact with the bacterial membrane and then disrupt the membrane structure. This work demonstrated a prospective approach for the design of antibacterial agents with highly effective antibacterial activity, high stability in plasma, and low cytotoxicity.

Preclinical study of a new 177Lu-labeled somatostatin receptor antagonist in HT-29 human colorectal cancer cells.

Somatostatin receptor-positive neuroendocrine tumors have been targeted using various peptide analogs radiolabeled with therapeutic radionuclides for years. The better biomedical properties of radioantagonists as higher tumor uptake make these radioligands more attractive than agonists for somatostatin receptor-targeted radionuclide therapy. In this study, we tried to evaluate the efficiency of Luthetium-177 (177Lu) radiolabeled DOTA-Peptide 2 (177Lu-DOTA-Peptide 2) as a new radioantagonist in HT-29 human colorectal cancer in vitro and in vivo. DOTA conjugated antagonistic peptide with the sequence of p-Cl-Phe-Cyclo(D-Cys-L-BzThi-D-Aph-Lys-Thr-Cys)-D-Tyr-NH2 (DOTA-Peptide 2) was labeled with 177Lu. In vitro assays (saturation binding assay and internalization test) and animal biodistribution were performed in human colon adenocarcinoma cells (HT-29) and HT-29 tumor-bearing nude mice. 177Lu-DOTA-Peptide 2 showed high stability in acetate buffer and human plasma (>97%). Antagonistic property of 177Lu-DOTA-Peptide 2 was confirmed by low internalization in HT-29 cells (<5%). The desired dissociation constant (Kd =11.14 nM) and effective tumor uptake (10.89 percentage of injected dose per gram of tumor) showed high binding affinity of 177Lu-DOTA-Peptide 2 to somatostatin receptors. 177Lu-DOTA-Peptide 2 demonstrated selective and high binding affinity to somatostatin receptors overexpressed on the surface of HT-29 cancer cells, which could make this radiopeptide suitable for somatostatin receptor-targeted radionuclide therapy.