Metallacarboranes in Medicinal Chemistry: Current Advances and Future Perspectives.

Metallacarboranes, exemplified by cobalt bis(dicarbollide) ([COSAN]-), have excelled their historical metallocene analogue label to become promising in drug design, medical studies, and fundamental biological research. Serving as a unique platform for conjugation with biomolecules, they also constitute an auspicious building block for biologically active derivatives and a carrier for cellular transport of membrane-impermeable cargos. Modified [COSAN]- exhibits specific antimicrobial, antiviral, and anticancer actions showing promise for preclinical trials. Contributing to the ongoing development in medicinal chemistry, metallacarboranes offer desirable physicochemical properties and low acute toxicity. This article presents a critical look at metallacarboranes in the context of their application in medicinal chemistry, emphasizing [COSAN]- as a potential game-changer in drug design and biomedical sciences. As medicinal chemistry seeks innovative building blocks, metallacarboranes emerge as an important novelty with versatile solutions and promising implications.

The modulation of local and systemic anti-tumor immune response induced by methotrexate nanoconjugate in murine MC38 colon carcinoma and B16 F0 melanoma tumor models.

Methotrexate (MTX) which is one of the longest-used cytostatics, belongs to the group of antimetabolites and is used for treatment in different types of cancer as well as during autoimmune diseases. MTX can act as a modulator enable to create the optimal environment to generate the specific anti-tumor immune response. A novel system for MTX delivery is its conjugation with high-molecular-weight carriers such as hydroxyethyl starch (HES), a modified amylopectin-based polymer applied in medicine as a colloidal plasma volume expander. Such modification prolongs the plasma half-life of the HES-MTX nanoconjugate and improves the distribution of the drug in the body. In the current study, we focused on evaluating the dose-dependent therapeutic efficacy of chemotherapy with HES-MTX nanoconjugate compared to the free form of MTX, and examining the time-dependent changes in the local and systemic anti-tumor immune response induced by this therapy. To confirm the higher effectiveness of HES-MTX in comparison to MTX, we analyzed its action using murine MC38 colon carcinoma and B16 F0 melanoma tumor models. It was noted that HES-MTX at a dose of 20 mg/kg b.w. was more effective in tumor growth inhibition than MTX in both tumor models. One of the main differences between the two analyzed tumor models concerned the kinetics of the appearance of the immunomodulation. In MC38 tumors, the beneficial change in the tumor microenvironment (TME) landscape, manifested by the depletion of pro-tumor immune cells, and increased influx of cells with strong anti-tumor activity was noted already 3 days after HES-MTX administration, while in B16 F0 model, these changes occurred 10 days after the start of therapy. Thus, the immunomodulatory potential of the HES-MTX nanoconjugate may be closely related to the specific immune cell composition of the TME, which combined with additional treatment such as immunotherapies, would enhance the therapeutic potential of the nanoconjugate.

Structural Patterns Enhancing the Antibacterial Activity of Metallacarborane-Based Antibiotics.

Healthcare systems heavily rely on antibiotics to treat bacterial infections, but the widespread presence of multidrug-resistant bacteria puts this strategy in danger. Novel drugs capable of overcoming current resistances are needed if our ability to treat bacterial infections is to be maintained. Boron clusters offer a valuable possibility to create a new class of antibiotics and expand the chemical space of antibiotics beyond conventional carbon-based molecules. In this work, we identified two promising structural patterns providing cobalta bis(dicarbollide)(COSAN)-based compounds with potent and selective activity toward Staphylococcus aureus (including clinical strains): introduction of the α-amino acid amide and addition of iodine directly to the metallacarborane cage. Furthermore, we found that proper hydrophilic-lipophilic balance is crucial for the selective activity of the tested compounds toward S. aureus over mammalian cells. The patterns proposed in this paper can be useful in the development of metallacarborane-based antibiotics with potent antibacterial properties and low cytotoxicity.

Evolution of the T4 phage virion is driven by selection pressure from non-bacterial factors.

Bacteriophages colonize animal and human bodies, propagating on sensitive bacteria that are symbionts, commensals, or pathogens of animals and humans. T4-like phages are dependent on abundant symbionts such as Escherichia coli, commonly present in animal and human gastrointestinal (GI) tracts. Bacteriophage T4 is one of the most complex viruses, and its intricate structure, particularly the capsid head protecting the phage genome, likely contributes substantially to the overall phage fitness in diverse environments. We investigated how individual head proteins-gp24, Hoc, and Soc-affect T4 phage survival under pressure from non-bacterial factors. We constructed a panel of T4 phage variants defective in these structural proteins: T4∆Soc, T4∆24byp24, T4∆Hoc∆Soc, T4∆Hoc∆24byp24, T4∆Soc∆24byp24, and T4∆Hoc∆Soc∆24byp24 (byp = bypass). These variants were investigated for their sensitivity to selected environmental conditions relevant to the microenvironment of the GI tract, including pH, temperature, and digestive enzymes. The simple and "primitive" structure of the phage capsid (∆24byp24) was significantly less stable at low pH and more sensitive to inactivation by digestive enzymes, and the simultaneous lack of gp24 and Soc resulted in a notable decrease in phage activity at 37°C. Gp24 was also found to be highly resistant to thermal and chemical denaturation. Thus, gp24, which was acquired relatively late in evolution, seems to play a key role in T4 withstanding environmental conditions, including those related to the animal/human GI tract, and Soc is a molecular glue that enhances this protective effect. IMPORTANCE Bacteriophages are important components of animal and human microbiota, particularly in the gastrointestinal tract, where they dominate the viral community and contribute to shaping microbial balance. However, interactions with bacterial hosts are not the only element of the equation in phage survival-phages inhabiting the GI tract are constantly exposed to increased temperature, pH fluctuations, or digestive enzymes, which raises the question of whether and how the complex structure of phage capsids contributes to their persistence in the specific microenvironment of human/animal bodies. Here we address this phage-centric perspective, identifying the role of individual head proteins in T4 phage survival in GI tract conditions. The selection pressure driving the evolution of T4-like phages could have come from the external environment that affects phage virions with increased temperature and variable pH; it is possible that in the local microenvironment along the GI tract, the phage benefits from stability-protecting proteins.

Inhibition of MC38 colon cancer growth by multicomponent chemoimmunotherapy with anti-IL-10R antibodies, HES-MTX nanoconjugate, depends on application of IL-12, IL-15 or IL-18 secreting dendritic cell vaccines.

Background: The tumor microenvironment (TME) provides a conducive environment for the growth and survival of tumors. Negative factors present in TME, such as IL-10, may limit the effectiveness of cellular vaccines based on dendritic cells, therefore, it is important to control its effect. The influence of IL-10 on immune cells can be abolished e.g., by using antibodies against the receptor for this cytokine - anti-IL-10R. Furthermore, the anticancer activity of cellular vaccines can be enhanced by modifying them to produce proinflammatory cytokines, such as IL-12, IL-15 or IL-18. Additionally, an immunomodulatory dose of methotrexate and hydroxyethyl starch (HES-MTX) nanoconjugate may stimulate effector immune cells and eliminate regulatory T cells, which should enhance the antitumor action of immunotherapy based on DC vaccines. The main aim of our study was to determine whether the HES-MTX administered before immunotherapy with anti-IL-10R antibodies would change the effect of vaccines based on dendritic cells overproducing IL-12, IL-15, or IL-18. Methods: The activity of modified DCs was checked in two therapeutic protocols - immunotherapy with the addition of anti-IL10R antibodies and chemoimmunotherapy with HES-MTX and anti-IL10R antibodies. The inhibition of tumor growth and the effectiveness of the therapy in inducing a specific antitumor response were determined by analyzing lymphoid and myeloid cell populations in tumor nodules, and the activity of restimulated splenocytes. Results and conclusions: Using the HES-MTX nanoconjugate before immunotherapy based on multiple administrations of anti-IL-10R antibodies and cellular vaccines capable of overproducing proinflammatory cytokines IL-12, IL-15 or IL-18 created optimal conditions for the effective action of these vaccines in murine colon carcinoma MC38 model. The applied chemoimmunotherapy caused the highest inhibition of tumor growth in the group receiving DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg at the level of 72.4%. The use of cellular vaccines resulted in cytotoxic activity increase in both immuno- or chemoimmunotherapy. However, the greatest potential was observed both in tumor tissue and splenocytes obtained from mice receiving two- or three-component vaccines in the course of combined application. Thus, the designed treatment schedule may be promising in anticancer therapy.

Cobalt bis(dicarbollide) is a DNA-neutral pharmacophore.

Cobalt bis(dicarbollide) (COSAN) is a metallacarborane used as a versatile pharmacophore to prepare biologically active hybrid organic-inorganic compounds or to improve the pharmacological properties of nucleosides, antisense oligonucleotides, and DNA intercalators. Despite these applications, COSAN interactions with nucleic acids remain unclear, limiting further advances in metallacarborane-based drug development. Although some studies showed that COSAN intercalates into DNA, COSAN-containing intercalators do not, and while COSAN shows low cytotoxicity, intercalators are often highly toxic. The present study aimed at comprehensively characterizing interactions between COSAN and DNA using a wide range of techniques, including UV-Vis absorption, circular (CD) and linear (LD) dichroism, nuclear magnetic resonance (NMR) spectroscopy, thermal denaturation, viscosity, differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC), and equilibrium dialysis measurements. Our results showed that COSAN has no effect on DNA structure, length, stability, or hybridization, with no or only faint signs of COSAN binding to DNA. Moreover, DNA is not necessary for COSAN to induce cytotoxicity at high concentrations, as shown by in vitro experiments. These findings demonstrate that COSAN is a DNA-neutral pharmacophore, thus confirming the general safety and biocompatibility of metallacarboranes and opening up new opportunities for further developing metallacarborane-based drugs.

Macro-GH - A clinical entity causing a diagnostic challenge - A case report.

AIM: Presentation of a new case of a patient with macro-GH, that may interfere with different GH assays leading to false-positive results in serum samples. CASE PRESENTATION: A 61-year-old female was referred with a pituitary macroadenoma and elevated growth hormone levels. The laboratory tests showed increased fasting GH level, measured by a sandwich chemiluminescence immunoassay (LIAISON® XL) without suppression on oral glucose tolerance test and normal IGF-1. The patient did not have the typical signs and symptoms of acromegaly. The patient underwent a transsphenoidal resection of a pituitary tumor, showing only α-subunit immunostaining. Postoperative GH levels remained elevated. An interference in the determination of GH level was suspected. GH was analyzed by three different immunoassays, UniCel DxI 600, Cobas e411 and hGH-IRMA. Heterophilic antibodies and rheumatoid factor were not detected in serum sample. GH recovery after precipitation with 25 % polyethylene glycol (PEG) was 12 %. Size-exclusion chromatography confirmed the presence of macro-GH in serum sample. CONCLUSION: If results of laboratory tests are not consistent with the clinical findings, the presence of an interference within immunochemical assays could be suspected. To identify interference caused by the macro-GH, the PEG method and size-exclusion chromatography should be used.

Combined therapy with methotrexate nanoconjugate and dendritic cells with downregulated IL-10R expression modulates the tumor microenvironment and enhances the systemic anti-tumor immune response in MC38 murine colon carcinoma.

Background: Understanding the negative impact of the tumor microenvironment on the creation of an effective immune response has contributed to the development of new therapeutic anti-cancer strategies. One such solution is combined therapy consisting of chemotherapeutic administration followed by dendritic cell (DC)-based vaccines. The use of cytostatic leads to the elimination of cancer cells, but can also modulate the tumor milieu. Moreover, great efforts are being made to increase the therapeutic outcome of immunotherapy, e.g. by enhancing the ability of DCs to generate an efficient immune response, even in the presence of immunosuppressive cytokines such as IL-10. The study aimed to determine the effectiveness of combined therapy with chemotherapeutic with immunomodulatory potential - HES-MTX nanoconjugate (composed of methotrexate (MTX) and hydroxyethyl starch (HES)) and DCs with downregulated expression of IL-10 receptor stimulated with tumor antigens (DC/shIL-10R/TAg) applied in MC38 murine colon carcinoma model. Methods: With the use of lentiviral vectors the DCs with decreased expression of IL-10R were obtained and characterized. During in vivo studies MC38-tumor bearing mice received MTX or HES-MTX nanoconjugate as a sole treatment or combined with DC-based immunotherapy containing unmodified DCs or DCs transduced with shRNA against IL-10R (or control shRNA sequence). Tumor volume was monitored during the experiment. One week after the last injection of DC-based vaccines, tumor nodules and spleens were dissected for ex vivo analysis. The changes in the local and systemic anti-tumor immune response were estimated with the use of flow cytometry and ELISA methods. Results and conclusions: In vitro studies showed that the downregulation of IL-10R expression in DCs enhances their ability to activate the specific anti-tumor immune response. The use of HES-MTX nanoconjugate and DC/shIL-10R/TAg in the therapy of MC38-tumor bearing mice resulted in the greatest tumor growth inhibition. At the local anti-tumor immune response level a decrease in the infiltration of cells with suppressor activity and an increase in the influx of effector cells into MC38 tumor tissue was observed. These changes were crucial to enhance the effective specific immune response at the systemic level, which was revealed in the greatest cytotoxic activity of spleen cells against MC38 cells.

Effect of Ovocystatin on Amyloid ß 1-42 Aggregation-In Vitro Studies.

Amyloid ß peptides (Aß) aggregating in the brain have a potential neurotoxic effect and are believed to be a major cause of Alzheimer's disease (AD) development. Thus, inhibiting amyloid polypeptide aggregation seems to be a promising approach to the therapy and prevention of this neurodegenerative disease. The research presented here is directed at the determination of the inhibitory activity of ovocystatin, the cysteine protease inhibitor isolated from egg white, on Aß42 fibril genesis in vitro. Thioflavin-T (ThT) assays, which determine the degree of aggregation of amyloid peptides based on fluorescence measurement, circular dichroism spectroscopy (CD), and transmission electron microscopy (TEM) have been used to assess the inhibition of amyloid fibril formation by ovocystatin. Amyloid beta 42 oligomer toxicity was measured using the MTT test. The results have shown that ovocystatin possesses Aß42 anti-aggregation activity and inhibits Aß42 oligomer toxicity in PC12 cells. The results of this work may help in the development of potential substances able to prevent or delay the process of beta-amyloid aggregation-one of the main reasons for Alzheimer's disease.

Metallacarborane Derivatives as Innovative Anti-Candida albicans Agents.

Infections caused by Candida species have increased significantly in the past decades and are among the leading causes of morbidity and mortality worldwide, resulting in serious public health problems. Currently, conventional antifungals are often ineffective as Candida spp. have developed growing resistance to systemic drugs. Since inorganic metallacarboranes are known to affect cellular events, new derivatives of these abiotic compounds were tested against Candida albicans. Compounds based on cobalt bis-dicarbollide [COSAN] were studied on Candida albicans strains, including a panel of 100 clinical isolates. The presented data prove that metallacarborane derivatives are effective against clinical isolates of Candida albicans, even those resistant to systemic drugs, and show synergistic potential in combination with amphotericin B, and low toxicity against human cells and Danio rerio embryos. This paper is a consequential step in the investigations of the broad spectrum and valuable future medical applications of metallacarboranes, especially in the fight against drug-resistant pathogens.

An alpha II spectrin mutant peptide with unstable scaffold structure and increased sensitivity to calpain cleavage.

A spontaneous missense mutation in the alpha II spectrin (αII) gene, replacing a highly conserved arginine 1098 with the glutamine (R1098Q), causes progressive neurodegeneration in heterozygous mutant mice. The molecular mechanism underlying this phenotype is unknown but the accumulation of 150kD αII breakdown products in brains of homozygous mutant embryos suggests an imbalance in the substrate level control of αII cleavage by calpains. This is further supported by in silico simulation predicting unmasked calpain target site and increased spectrin scaffold bending and flexibility of R1098Q mutant peptide. Here, using spectroscopic and in situ enzymatic techniques, we aimed at obtaining direct experimental support for the impact of R1098Q mutation on the αII stability and its propensity for calpain-mediated degradation. Thermal circular dichroism analyses performed on recombinant wildtype and R1098Q mutant αII peptides, composed of spectrin repeat 9-10 revealed that although both had very similar secondary structure contents, thermal stability curve profiles varied and the observed midpoint of the unfolding transition (Tm) was 5.5 °C lower for the R1098Q peptide. Yet, the dynamic light scattering profiles of both peptides closely overlapped, implying the same thermal propensity to aggregate. Calpain digestion of plate-bound αII peptides with and without added calmodulin revealed an enhancement of the R1098Q peptide digestion rate relative to WT control. In summary, these results support the unstable scaffold structure of the R1098Q peptide as contributing to its enhanced intrinsic sensitivity to calpain and suggest physiologic relevance of a proper calpain/spectrin balance in preventing neurodegeneration.

Aromatic Bis[aminomethylidenebis(phosphonic)] Acids Prevent Ovariectomy-Induced Bone Loss and Suppress Osteoclastogenesis in Mice.

Osteoporosis is a skeletal disease associated with excessive bone turnover. Among the compounds with antiresorptive activity, nitrogen-containing bisphosphonates play the most important role in antiosteoporotic treatment. In previous studies, we obtained two aminomethylidenebisphosphonates-benzene-1,4-bis[aminomethylidene(bisphosphonic)] (WG12399C) acid and naphthalene-1,5-bis[aminomethylidene(bisphosphonic)] (WG12592A) acid-which showed a significant antiproliferative activity toward J774E macrophages, a model of osteoclast precursors. The aim of these studies was to evaluate the antiresorptive activity of these aminobisphosphonates in ovariectomized (OVX) Balb/c mice. The influence of WG12399C and WG12592A administration on bone microstructure and bone strength was studied. Intravenous injections of WG12399C and WG12592A bisphosphonates remarkably prevented OVX-induced bone loss; for example, they sustained bone mineral density at control levels and restored other bone parameters such as trabecular separation. This was accompanied by a remarkable reduction in the number of TRAP-positive cells in bone tissue. However, a significant improvement in the quality of bone structure did not correlate with a parallel increase in bone strength. In ex vivo studies, WG12399C and WG12592A remarkably bisphosphonates reduced osteoclastogenesis and partially inhibited the resorptive activity of mature osteoclasts. Our results show interesting biological activity of two aminobisphosphonates, which may be of interest in the context of antiresorptive therapy.

Solid-State, Thermal Synthesis of Peptide/Protein-Boron Cluster Conjugates.

Anionic boron clusters can be used to increase the pharmaceutical properties of the peptides. Here, we describe the method of synthesis of peptide/protein-boron cluster conjugates using solid-state, thermal reaction on two different peptides: thymosin ß4 (Tß4) and lysozyme. 1,4-dioxane oxonium derivatives of anionic boron clusters are used as donors of boron clusters. This procedure allows to conjugate anionic boron clusters to native peptides without loss of the activity of the peptides.

Metallacarborane Derivatives Effective against Pseudomonas aeruginosa and Yersinia enterocolitica.

Pseudomonas aeruginosa is an opportunistic human pathogen that has become a nosocomial health problem worldwide. The pathogen has multiple drug removal and virulence secretion systems, is resistant to many antibiotics, and there is no commercial vaccine against it. Yersinia pestis is a zoonotic pathogen that is on the Select Agents list. The bacterium is the deadliest pathogen known to humans and antibiotic-resistant strains are appearing naturally. There is no commercial vaccine against the pathogen, either. In the current work, novel compounds based on metallacarborane cage were studied on strains of Pseudomonas aeruginosa and a Yersinia pestis substitute, Yersinia enterocolitica. The representative compounds had IC50 values below 10 µM against Y. enterocolitica and values of 20-50 µM against P. aeruginosa. Artificial generation of compound-resistant Y. enterocolitica suggested a common mechanism for drug resistance, the first reported in the literature, and suggested N-linked metallacarboranes as impervious to cellular mechanisms of resistance generation. SEM analysis of the compound-resistant strains showed that the compounds had a predominantly bacteriostatic effect and blocked bacterial cell division in Y. enterocolitica. The compounds could be a starting point towards novel anti-Yersinia drugs and the strategy presented here proposes a mechanism to bypass any future drug resistance in bacteria.

Comparison of Antibacterial Mode of Action of Silver Ions and Silver Nanoformulations With Different Physico-Chemical Properties: Experimental and Computational Studies.

The aim of this study was to compare the antibacterial mode of action of silver ions (Ag+) and selected silver nanoformulations against E. coli strains (E. coli J53, Escherichia coli BW25113 and its derivatives: Δ ompA, Δ ompC, Δ ompF, Δ ompR, ompRG596AcusSG1130A, cusSG1130A). In this research we used various experimental methods and techniques such as determination of the minimal inhibitory concentration, flow cytometry, scanning electron microscopy, circular dichroism as well as computational methods of theoretical chemistry. Thanks to the processing of bacteria and silver samples (ions and nanoformulations), we were able to determine the bacterial sensitivity to silver samples, detect reactive oxygen species (ROS) in the bacterial cells, visualize the interaction of silver samples with the bacterial cells, and identify their interactions with proteins. Differences between the mode of action of silver ions and nanoformulations and the action of nanoformulations themselves were revealed. Based on the results of computational methods, we proposed an explanation of the differences in silver-outer protein interaction between silver ions and metallic silver; in general, the Ag0 complexes exhibit weaker interaction than Ag+ ones. Moreover, we identified two gutter-like areas of the inner layer of the ion channel: one more effective, with oxygen-rich side chains; and another one less effective, with nitrogen-rich side chains.

Age-dependent ataxia and neurodegeneration caused by an αII spectrin mutation with impaired regulation of its calpain sensitivity.

The neuronal membrane-associated periodic spectrin skeleton (MPS) contributes to neuronal development, remodeling, and organization. Post-translational modifications impinge on spectrin, the major component of the MPS, but their role remains poorly understood. One modification targeting spectrin is cleavage by calpains, a family of calcium-activated proteases. Spectrin cleavage is regulated by activated calpain, but also by the calcium-dependent binding of calmodulin (CaM) to spectrin. The physiologic significance of this balance between calpain activation and substrate-level regulation of spectrin cleavage is unknown. We report a strain of C57BL/6J mice harboring a single αII spectrin point mutation (Sptan1 c.3293G > A:p.R1098Q) with reduced CaM affinity and intrinsically enhanced sensitivity to calpain proteolysis. Homozygotes are embryonic lethal. Newborn heterozygotes of either gender appear normal, but soon develop a progressive ataxia characterized biochemically by accelerated calpain-mediated spectrin cleavage and morphologically by disruption of axonal and dendritic integrity and global neurodegeneration. Molecular modeling predicts unconstrained exposure of the mutant spectrin's calpain-cleavage site. These results reveal the critical importance of substrate-level regulation of spectrin cleavage for the maintenance of neuronal integrity. Given that excessive activation of calpain proteases is a common feature of neurodegenerative disease and traumatic encephalopathy, we propose that damage to the spectrin MPS may contribute to the neuropathology of many disorders.

Immunomodulatory potential of anticancer therapy composed of methotrexate nanoconjugate and dendritic cell­based vaccines in murine colon carcinoma.

Chemotherapy with low­molecular weight compounds, despite elimination of cancer cells, entails adverse effects. To overcome this disadvantage, innovative drug delivery systems are being developed, including conjugation of macromolecular carriers with therapeutics, e.g. a nanoconjugate of hydroxyethyl starch and methotrexate (HES­MTX). The purpose of the present study was to determine whether HES­MTX, applied as a chemotherapeutic, is able to modulate the immune response and support the antitumor response generated by dendritic cells (DCs) used subsequently as immunotherapeutic vaccines. Therefore, MTX or HES­MTX was administered, as sole treatment or combined with DC­based vaccines, to MC38 colon carcinoma tumor­bearing mice. Alterations in antitumor immune response were evaluated by multiparameter flow cytometry analyses and functional assays. The results demonstrated that the nanoconjugate possesses greater immunomodulatory potential than MTX as reflected by changes in the landscape of immune cells infiltrating the tumor and increased cytotoxicity of splenic lymphocytes. In contrast to MTX, therapy with HES­MTX as sole treatment or combined with DC­based vaccines, contributed to significant tumor growth inhibition. However, only treatment with HES­MTX and DC­based vaccines activated the systemic specific antitumor response. In conclusion, due to its immunomodulatory properties, the HES­MTX nanoconjugate could become a potent anticancer agent used in both chemo­ and chemoimmunotherapeutic treatment schemes.

Metallacarboranes as a tool for enhancing the activity of therapeutic peptides.

Metallacarboranes are anionic boron clusters with high affinity to serum albumin, ability to cross biological membranes, and no apparent toxicity in vitro and in vivo. Thus, conjugation with cobalt bis(1,2-dicarbollide), [COSAN]- , ([3,3'-Co(1,2-C2 B9 H11 )2 ]- ) may improve the properties of therapeutic peptides or proteins at both molecular and systemic levels. Here, we conjugated [COSAN]- with the therapeutic peptide thymosin ß4 (Tß4), which has a pleiotropic activity that results in enhanced healing and regeneration of injured tissues. Using fluorescence quenching of human serum albumin and surface plasmon resonance techniques, we showed that the conjugates have a high affinity to human serum albumin. Using an in vitro wound closure assay, we showed that conjugation with [COSAN]- enhances the activity of Tß4 toward fibroblasts (MSU1.1 cell line). These results indicate an application of metallacarboranes in the development of analogs of various therapeutic peptides/proteins with superior pharmacological properties.

Temporal inhibition of mouse mammary gland cancer metastasis by CORM-A1 and DETA/NO combination therapy.

Carbon monoxide and nitric oxide are two of the most important vasoprotective mediators. Their downregulation observed during vascular dysfunction, which is associated with cancer progression, leads to uncontrolled platelet activation. Therefore, the aim of our studies was to improve vasoprotection and to decrease platelet activation during progression of mouse mammary gland cancer by concurrent use of CO and NO donors (CORM-A1 and DETA/NO, respectively). Methods: Mice injected intravenously with 4T1-luc2-tdTomato or orthotopically with 4T1 mouse mammary gland cancer cells were treated with CORM-A1 and DETA/NO. Ex vivo aggregation and activation of platelets were assessed in the blood of healthy donors and breast cancer patients. Moreover, we analyzed the compounds' direct effect on 4T1 mouse and MDA-MB-231 human breast cancer cells proliferation, adhesion and migration in vitro. Results: We have observed antimetastatic effect of combination therapy, which was only transient in orthotopic model. During early stages of tumor progression concurrent use of CORM-A1 and DETA/NO demonstrated vasoprotective ability (decreased endothelin-1, sICAM and sE-selectin plasma level) and downregulated platelets activation (decreased bound of fibrinogen and vWf to platelets) as well as inhibited EMT process. Combined treatment with CO and NO donors diminished adhesion and migration of breast cancer cells in vitro and inhibited aggregation as well as TGF-ß release from breast cancer patients' platelets ex vivo. However, antimetastatic effect was not observed at a later stage of tumor progression which was accompanied by increased platelets activation and endothelial dysfunction related to a decrease of VASP level. Conclusion: The therapy was shown to have antimetastatic action and resulted in normalization of endothelial metabolism, diminution of platelet activation and inhibition of EMT process. The effect was more prominent during early stages of tumor dissemination. Such treatment could be applied to inhibit metastasis during the first stages of this process.