ABSTRACT
Cancer treatment with vascular disrupting agents (VDAs) causes rapid and extensive necrosis in solid tumors. However, these agents fall short in eliminating all malignant cells, ultimately leading to tumor regrowth. Here, we investigated whether the molecular changes in the tumor microenvironment induced by VDA treatment sensitize the tumors for secondary nanotherapy enhanced by clinical-stage tumor penetrating peptide iRGD. Treatment of peritoneal carcinomatosis (PC) and breast cancer mice with VDA combretastatin A-4 phosphate (CA4P) resulted in upregulation of the iRGD receptors αv-integrins and NRP-1, particularly in the peripheral tumor tissue. In PC mice treated with CA4P, coadministration of iRGD resulted in an approximately threefold increase in tumor accumulation and a more homogenous distribution of intraperitoneally administered nanoparticles. Notably, treatment with a combination of CA4P, iRGD, and polymersomes loaded with a novel anthracycline Utorubicin (UTO-PS) resulted in a significant decrease in the overall tumor burden in PC-bearing mice, while avoiding overt toxicities. Our results indicate that VDA-treated tumors can be targeted therapeutically using iRGD-potentiated nanotherapy and warrant further studies on the sequential targeting of VDA-induced molecular signatures.
Subject(s)
Nanoparticles , Tumor Microenvironment , Animals , Tumor Microenvironment/drug effects , Mice , Female , Nanoparticles/chemistry , Bibenzyls/pharmacology , Bibenzyls/chemistry , Cell Line, Tumor , Humans , Stilbenes/pharmacology , Stilbenes/administration & dosage , Oligopeptides/chemistry , Oligopeptides/pharmacology , Neuropilin-1/metabolism , Peritoneal Neoplasms/drug therapy , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/administration & dosageABSTRACT
Novel anticancer compounds and their precision delivery systems are actively developed to create potent and well-tolerated anticancer therapeutics. Here, we report the synthesis of a novel anthracycline, Utorubicin (UTO), and its preclinical development as an anticancer payload for nanocarriers. Free UTO was significantly more toxic to cultured tumor cell lines than the clinically used anthracycline, doxorubicin. Nanoformulated UTO, encapsulated in polymeric nanovesicles (polymersomes, PS), reduced the viability of cultured malignant cells and this effect was potentiated by functionalization with a tumor-penetrating peptide (TPP). Systemic peptide-guided PS showed preferential accumulation in triple-negative breast tumor xenografts implanted in mice. At the same systemic UTO dose, the highest UTO accumulation in tumor tissue was seen for the TPP-targeted PS, followed by nontargeted PS, and free doxorubicin. Our study suggests potential applications for UTO in the treatment of malignant diseases and encourages further preclinical and clinical studies on UTO as a nanocarrier payload for precision cancer therapy.
Subject(s)
Antineoplastic Agents/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Mammary Neoplasms, Experimental/diagnostic imaging , Mammary Neoplasms, Experimental/drug therapy , Mice , Molecular Conformation , Optical Imaging , Structure-Activity RelationshipABSTRACT
Novel anticancer compounds and their precision delivery systems are actively developed to create potent and well-tolerated anticancer therapeutics. Here, we report the synthesis of a novel anthracycline, Utorubicin (UTO), and its preclinical development as an anticancer payload for nanocarriers. Free UTO was significantly more toxic to cultured tumor cell lines than the clinically used anthracycline, doxorubicin. Nanoformulated UTO, encapsulated in polymeric nanovesicles (polymersomes, PS), reduced the viability of cultured malignant cells and this effect was potentiated by functionalization with a tumor-penetrating peptide (TPP). Systemic peptide-guided PS showed preferential accumulation in triple-negative breast tumor xenografts implanted in mice. At the same systemic UTO dose, the highest UTO accumulation in tumor tissue was seen for the TPP-targeted PS, followed by nontargeted PS, and free doxorubicin. Our study suggests potential applications for UTO in the treatment of malignant diseases and encourages further preclinical and clinical studies on UTO as a nanocarrier payload for precision cancer therapy.
ABSTRACT
A novel set of 1-substituted apomorphines as dopaminergic agonists were synthesized according to our new strategy employing the acid-catalyzed rearrangement of diversely functionalized 5ß-substituted-6-demethoxythebaines. The activities of new compounds for dopamine receptors subtypes were evaluated using HEK293 based stable cell lines expressing D1, D2L or D3 receptor subtypes. All studied compounds had affinities in nanomolar range for D2L and D3 receptors and the change of the nature of substituent in position 1 had only moderate effect. D1 receptors were sensitive to the introduction of the 4-OH-benzyl function resulting in an increased affinity. The small hydrophilic group (hydroxymethyl) highly reduced the agonist affinity and potency thereby increasing subtype selectivity. This strategy for selective modulation of affinities and potencies of 1-substituted apomorphines gives essential hints for future design of subtype selective dopaminergic ligands.
Subject(s)
Apomorphine/chemical synthesis , Apomorphine/pharmacology , Dopamine Agonists/chemical synthesis , Dopamine Agonists/pharmacology , Drug Design , Apomorphine/chemistry , Binding Sites , Dopamine Agonists/chemistry , HEK293 Cells , Humans , Molecular Structure , Protein Binding/drug effects , Receptors, Dopamine/metabolism , Structure-Activity RelationshipABSTRACT
We have synthesized novel 2-O-substituted apomorphines with both different lengths of lipophilic alkyl chains and alkyl chains carrying free hydroxyl groups. Two bis-apomorphines formed as side products of the reactions with diols were isolated and characterized as well. The neuropharmacological profile of all these new compounds were investigated with respect to their binding affinities and activities to dopamine D(2) and D(1) receptors. The obtained data pointed to the fact that, in the examination of dopaminergic activities of 2-substituted apomorphines, the lipophilicity of the substituent is more important than its spatial parameters.
Subject(s)
Apomorphine/chemical synthesis , Apomorphine/pharmacology , Neurons/drug effects , Animals , Apomorphine/chemistry , CHO Cells , Catalysis , Cricetinae , Cricetulus , Models, Molecular , Molecular Structure , Receptors, Dopamine D1/metabolism , Receptors, Dopamine D2/metabolism , Structure-Activity RelationshipABSTRACT
Serotonin (5-HT) system has a significant role in anxiety- and depression-related states and may be influenced by brain-derived neurotrophic factor (BDNF). This study examined extracellular 5-HT levels and expression of BDNF in rats with persistently low or high levels of exploratory activity (LE and HE, respectively). Baseline extracellular levels of 5-HT as assessed by in vivo microdialysis in conscious animals were similar in both groups in medial prefrontal cortex (PFC) and dentate gyrus (DG). No differences were found in parachloroamphetamine-induced 5-HT release in either region. However, LE animals had significantly higher levels of 5-HT transporter (5-HTT) binding in PFC and a larger increase in extracellular 5-HT levels after administration of citalopram (1 microM) into this area by retrograde dialysis. No difference in 5-HTT levels was found in hippocampus, while perfusion with citalopram was accompanied by a greater increase in extracellular 5-HT in the HE group in this brain region. LE-rats had higher levels of BDNF mRNA in the PFC but not hippocampus. In contrast, levels of nerve growth factor mRNA were similar in these brain regions of LE- and HE-rats. The differential regulation of 5-HT-ergic system in LE- and HE-rats in PFC and hippocampus may form the basis for their distinct anxiety-related behaviours.
Subject(s)
Brain-Derived Neurotrophic Factor/metabolism , Exploratory Behavior/physiology , Extracellular Fluid/metabolism , Gene Expression Regulation/physiology , Serotonin/metabolism , Analysis of Variance , Animals , Behavior, Animal/drug effects , Brain-Derived Neurotrophic Factor/genetics , Citalopram/pharmacology , Exploratory Behavior/drug effects , Gene Expression Regulation/drug effects , Male , Prefrontal Cortex/drug effects , Prefrontal Cortex/metabolism , RNA, Messenger/metabolism , Rats , Rats, Wistar , Serotonin Agents/pharmacology , Selective Serotonin Reuptake Inhibitors/pharmacology , p-Chloroamphetamine/pharmacologyABSTRACT
There are several evidences that some functions of D1 dopamine receptors can be modulated by colocalized adenosine A1 receptors. To elucidate the role of particular components of the receptor complex in the ligand binding and second messenger activation level we have used Sf9 cell expression system. The expression of D1 and A1 receptors was confirmed by proper binding of specific radioligands [3H]SCH23390 (Kd=1.1+/-0.1 nM, Bmax=2.2+/-0.1 pmol/mg protein) and [3H]DPCPX (Kd=2.1+/-0.8nM, Bmax=2.9+/-0.4 pmol/mg protein), respectively. The kinetics of [3H]SCH23390 binding corresponded to the simplest reversible bimolecular binding reaction of complex formation, with k(on)=0.20+/-0.02 min(-1)nM(-1) and k(off)=0.13+/-0.01 min(-1). Dopaminergic agonists increased the accumulation of cAMP in the transfected cells in concentration-dependent manner, indicating a correct coupling of receptor to second messenger system. The coupling of the A1 receptor to Gi proteins was confirmed by both GTPgammaS dependent agonist binding and inhibition of cAMP accumulation by N-cyclopentyladenosine (NCPA). Activation of the A1 receptor by NCPA had no significant influence on neither affinities of dopaminergic ligands nor the radioligand binding kinetics to the co-exprssed D1 receptors in Sf9 cell membranes. On the other hand, the activation of the A1 receptors inhibited the D1 receptor-specific accumulation of cAMP, but only in cells where Gi proteins were expressed with the receptors.
Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Gene Expression/physiology , Receptor, Adenosine A1/physiology , Receptors, Dopamine D1/physiology , Signal Transduction/physiology , 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine/pharmacology , Adenosine/analogs & derivatives , Adenosine/pharmacology , Adenosine A1 Receptor Antagonists , Animals , Benzazepines/pharmacokinetics , Binding, Competitive/drug effects , Cell Line , Cyclic AMP/metabolism , Dopamine Agonists/pharmacology , Dopamine Antagonists/pharmacokinetics , Dose-Response Relationship, Drug , Drug Interactions , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Gene Expression/drug effects , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , Humans , Radioligand Assay/methods , Spodoptera , Transfection/methods , Tritium/pharmacokinetics , Xanthines/pharmacokineticsABSTRACT
We have characterized the binding of [2-(3)H]-4-(2-[7-Amino-2-(2-furyl)-[1,2,4]-triazolo-[2,3-a]-[1,3,5]-triazin-5-ylamino]ethyl)phenol ([(3)H]ZM241385) to adenosine A(2A) receptors in membranes of rat striatum and transfected CHO cells. Saturation experiments showed that [(3)H]ZM241385 binds to a single class of binding sites with high affinity (K(d) = 0.23 nM and 0.14 nM in CHO cell and striatal membranes, respectively). The membranes of CHO cells required pretreatment with adenosine deaminase (ADA) to achieve high-affinity binding, while ADA had no influence on the ligand binding properties in striatal membranes. The binding of [(3)H]ZM241385 was fast and reversible, achieving equilibrium within 20 minutes at all radioligand concentrations. The kinetic analysis of the [(3)H]ZM241385 interaction with A(2A) receptors indicated that the reaction had at least two subsequent steps. The first step corresponds to a fast equilibrium, which also determines the antagonist potency to competitively inhibit CGS21680-induced accumulation of cAMP (first equilibrium constant K(A) = 6.6 nM). The second step corresponds to a slow process of conformational isomerization (equilibrium constant K(i) = 0.03). The combination of the two steps gives the dissociation constant K(d) = 0.20 nM based on the kinetic data, which is in good agreement with the directly measured value. The data obtained shed light on the mechanism of the [(3)H]ZM241385 interaction with adenosine A(2A) receptors from different sources in vitro. The isomerization step of the A(2A) antagonist radioligand binding has to be taken into account for the interpretation of the binding parameters obtained from the various competition assays and explain the discrepancy between antagonist affinity in saturation experiments versus its potency in functional assays.
Subject(s)
Adenosine A2 Receptor Antagonists , Adenosine/analogs & derivatives , Triazines/pharmacology , Triazoles/pharmacology , Adenosine/agonists , Adenosine/antagonists & inhibitors , Adenosine/metabolism , Algorithms , Animals , Binding, Competitive/drug effects , CHO Cells , Cell Membrane/drug effects , Cell Membrane/metabolism , Cricetinae , Cyclic AMP/metabolism , GTP-Binding Proteins/metabolism , Kinetics , Ligands , Neostriatum/drug effects , Neostriatum/metabolism , Phenethylamines/metabolism , Radioligand AssayABSTRACT
The M2 muscarinic acetylcholine receptor (mAChR) expressed in insect cells (Spodoptera frugiperda, Sf9) using the baculovirus system formed active functional complexes with coexpressed Gi as well as with Go proteins, while no complexes could be detected with internal G proteins. Comparison of the abilities of different muscarinic agonists and partial agonists to increase [35S]GTPgammaS binding revealed no significant differences between M2/Gi and M2/Go complexes neither with respect to affinities nor efficacies of the ligands studied. Coexpression with either G protein caused constitutive activity of the receptor amounting up to 66% of stimulable [35S]GTPgammaS binding. Muscarinic antagonists, like atropine, scopolamine and N-methylscopolamine, behaved as inverse antagonists with potencies in good agreement with their binding affinities to the receptor. The results implicate that the functional reconstitution of M2 muscarinic receptor with either Gi or Go proteins in insect cells provides a valuable tool for screening of potencies as well as efficacies of agonists, partial agonists and inverse agonists at this receptor.