ABSTRACT
The bromodomain and extra-terminal (BET) family of proteins, consisting of the bromodomains containing protein 2 (BRD2), BRD3, BRD4, and the testis-specific BRDT, are key epigenetic regulators of gene transcription and has emerged as an attractive target for anticancer therapy. Herein, we describe the discovery of a novel potent BET bromodomain inhibitor, using a systematic structure-based approach focused on improving potency, metabolic stability, and permeability. The optimized dimethylisoxazole aryl-benzimidazole inhibitor exhibited high potency towards BRD4 and related BET proteins in biochemical and cell-based assays and inhibited tumor growth in two proof-of-concept preclinical animal models.
Subject(s)
Benzimidazoles/pharmacology , Drug Discovery , Isoxazoles/pharmacology , Multiple Myeloma/drug therapy , Transcription Factors/antagonists & inhibitors , Administration, Oral , Animals , Benzimidazoles/chemistry , Benzimidazoles/metabolism , Biological Availability , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Humans , Isoxazoles/administration & dosage , Isoxazoles/chemistry , Isoxazoles/metabolism , Mice , Molecular Structure , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Protein Domains/drug effects , Structure-Activity Relationship , Transcription Factors/metabolismABSTRACT
The most recent Ebola virus outbreak in West Africa, which was unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected, highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae. No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we report the discovery of a novel small molecule GS-5734, a monophosphoramidate prodrug of an adenosine analogue, with antiviral activity against EBOV. GS-5734 exhibits antiviral activity against multiple variants of EBOV and other filoviruses in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternative substrate and RNA-chain terminator in primer-extension assays using a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life, 14 h) and distribution to sanctuary sites for viral replication including testes, eyes, and brain. In a rhesus monkey model of EVD, once-daily intravenous administration of 10 mg kg(-1) GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two out of six treated animals. These results show the first substantive post-exposure protection by a small-molecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses, including filoviruses, arenaviruses, and coronaviruses, suggests the potential for wider medical use. GS-5734 is amenable to large-scale manufacturing, and clinical studies investigating the drug safety and pharmacokinetics are ongoing.
Subject(s)
Alanine/analogs & derivatives , Antiviral Agents/therapeutic use , Hemorrhagic Fever, Ebola/drug therapy , Macaca mulatta/virology , Ribonucleotides/therapeutic use , Adenosine Monophosphate/analogs & derivatives , Alanine/pharmacokinetics , Alanine/pharmacology , Alanine/therapeutic use , Amino Acid Sequence , Animals , Antiviral Agents/pharmacokinetics , Antiviral Agents/pharmacology , Cell Line, Tumor , Ebolavirus/drug effects , Female , HeLa Cells , Hemorrhagic Fever, Ebola/prevention & control , Humans , Male , Molecular Sequence Data , Organ Specificity , Prodrugs/pharmacokinetics , Prodrugs/pharmacology , Prodrugs/therapeutic use , Ribonucleotides/pharmacokinetics , Ribonucleotides/pharmacologyABSTRACT
HPMA copolymer-RGDfK (HPMA-RGDfK) conjugates bearing either aminohexylgeldanamycin (AHGDM) or docetaxel (DOC) were synthesized and characterized. In vitro stability and binding were evaluated. Cytotoxicity toward ovarian cancer cells was evaluated and the ability of the conjugates to induce cell death was assessed by combination index analysis. Conjugates bearing AHGDM were more stable and exhibited slower drug release than those bearing DOC. Both conjugates demonstrated the ability to bind to avb3 integrins. In combination, HPMA-RGDfK conjugates demonstrated marked synergism as compared to their non-targeted counterparts and free drug controls. HPMA-RGDfK conjugates bearing AHGDM and DOC induce synergistic cytotoxicity in vitro, suggesting their potential as a promising combination therapy.
Subject(s)
Antibiotics, Antineoplastic , Benzoquinones , Lactams, Macrocyclic , Methacrylates , Oligopeptides , Ovarian Neoplasms/drug therapy , Taxoids , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/pharmacokinetics , Antibiotics, Antineoplastic/pharmacology , Benzoquinones/chemistry , Benzoquinones/pharmacokinetics , Benzoquinones/pharmacology , Cell Death/drug effects , Cell Line, Tumor , Docetaxel , Drug Screening Assays, Antitumor , Female , Humans , Integrin alphaVbeta3/antagonists & inhibitors , Lactams, Macrocyclic/chemistry , Lactams, Macrocyclic/pharmacokinetics , Lactams, Macrocyclic/pharmacology , Methacrylates/chemistry , Methacrylates/pharmacokinetics , Methacrylates/pharmacology , Oligopeptides/chemistry , Oligopeptides/pharmacokinetics , Oligopeptides/pharmacology , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Taxoids/chemistry , Taxoids/pharmacokinetics , Taxoids/pharmacologyABSTRACT
The synthesis, characterization, and in vitro evaluation of a combination delivery of multiblock poly(N-2-hydroxypropyl)methacrylamide (HPMA), gemcitabine (GEM) and paclitaxel (PTX) conjugates is described in this study. Multiblock copolymer conjugates of a large molecular weight (Mw>200 kDa) were studied and compared to traditional, small molecular weight (Mw<45 kDa) conjugates. Stability of the conjugates in different pH was assessed, and their cytotoxicity in combination toward A2780 human ovarian cancer cells was evaluated by combination index analysis. Treatment duration (4 and 72 h) and sequence of addition were explored. In addition, an HPMA copolymer conjugate with both GEM and PTX in the side chains was evaluated in a similar manner and compared to a physical mixture of individual conjugates. Conjugates with narrow molecular weight distribution (Mw/Mn<1.1) were obtained via RAFT polymerization, and drug loadings of between 5.5 and 9.2 wt% were achieved. Conjugates demonstrated moderate stability with less than 65% release over 24h at pH 7.4, and near complete drug release in the presence of the lysosomal enzyme cathepsin B in 3h. In combination, the cytotoxic effects of a mixture of the conjugates were primarily additive. Synergistic effects were observed when A2780 human ovarian cancer cells were treated simultaneously for 4h with multiblock conjugates (CI<0.7). When both GEM and PTX were conjugated to the same copolymer backbone, moderate antagonism (CI 1.3-1.6) was observed. These results demonstrate that multiblock HPMA copolymer-GEM and -PTX conjugates, when delivered as a mixture of individual agents, are promising for the treatment of ovarian cancer.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Deoxycytidine/analogs & derivatives , Drug Carriers , Ovarian Neoplasms/pathology , Paclitaxel/pharmacology , Polymethacrylic Acids/chemistry , Cathepsin B/chemistry , Cell Line, Tumor , Cell Survival/drug effects , Chemistry, Pharmaceutical , Deoxycytidine/administration & dosage , Deoxycytidine/chemistry , Deoxycytidine/pharmacology , Drug Stability , Drug Synergism , Female , Humans , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Male , Molecular Weight , Paclitaxel/administration & dosage , Paclitaxel/chemistry , Polymerization , Solubility , Technology, Pharmaceutical/methods , GemcitabineABSTRACT
In the field of nanomedicine, selective delivery to cancer cells is a common goal, where active targeting strategies are often employed to increase tumor accumulation. In this study, tumor hyperthermia was utilized as a means to increase the active delivery of heat shock protein (HSP) targeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-drug conjugates. Following hyperthermia, induced expression of cell surface heat shock protein (HSP) glucose regulated protein 78 kDa (GRP78) was utilized for targeted drug therapy. Conjugates bearing the anticancer agents aminohexylgeldanamycin (AHGDM), docetaxel (DOC), or cisplatin and the GRP78 targeting peptide WDLAWMFRLPVG were synthesized and characterized. Binding to cell surface expressed heat shock protein GRP78 on the surface of human prostate cancer DU145 cells was evaluated. HSP targeted AHGDM and DOC conjugates demonstrated active binding comparable to native targeting peptide. They were then assessed in vitro for the ability to synergistically induce cytotoxicity in combination with moderate hyperthermia (43 °C, 30 min). HSP targeted DOC conjugates exhibited high potency against DU145 cells with an IC50 of 2.4 nM. HSP targeted AHGDM and DOC conjugates demonstrated synergistic effects in combination with hyperthermia with combination index values of 0.65 and 0.45 respectively. Based on these results, HSP targeted DOC conjugates were selected for in vivo evaluation. In DU145 tumor bearing mice, a single treatment of tumor hyperthermia, induced via gold nanorod mediated plasmonic photothermal therapy, and intravenous administration of HSP targeted HPMA copolymer-docetaxel at 10mg/kg resulted in maintained tumor regression for a period of 30 days. These results demonstrate the potential for tumor hyperthermia to increase the delivery of HSP targeted macromolecular chemotherapeutics.
Subject(s)
Antineoplastic Agents/administration & dosage , Heat-Shock Proteins/metabolism , Hyperthermia, Induced , Neoplasms/therapy , Animals , Antineoplastic Agents/chemistry , Benzoquinones/administration & dosage , Benzoquinones/chemistry , Cell Line, Tumor , Cell Survival/drug effects , Cisplatin/administration & dosage , Cisplatin/chemistry , Combined Modality Therapy , Docetaxel , Endoplasmic Reticulum Chaperone BiP , Gold/chemistry , Humans , Lactams, Macrocyclic/administration & dosage , Lactams, Macrocyclic/chemistry , Methacrylates/chemistry , Mice , Mice, Nude , Nanotubes/chemistry , Neoplasms/metabolism , Oligopeptides/chemistry , Taxoids/administration & dosage , Taxoids/chemistryABSTRACT
Effective drug delivery to tumors requires both transport through the vasculature and tumor interstitium. Previously, it was shown that gold nanorod (GNR) mediated plasmonic photothermal therapy (PPTT) is capable of increasing the overall accumulation of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers in prostate tumors. In the present study, it is demonstrated that PPTT is also capable of increasing the distribution of these conjugates in tumors. Gadolinium labeled HPMA copolymers were administered to mice bearing prostate tumors immediately before treatment of the right tumor with PPTT. The left tumor served as internal, untreated control. Magnetic resonance imaging (MRI) of both tumors showed that PPTT was capable of improving the tumor mass penetration of HPMA copolymers. Thermal enhancement of delivery, roughly 1.5-fold, to both the tumor center and periphery was observed. Confocal microscopy of fluorescently labeled copolymers corroborates these findings in that PPTT is capable of delivering more HPMA copolymers to the tumor's center and periphery. These results further demonstrate that PPTT is a useful tool to improve the delivery of polymer-drug conjugates.
Subject(s)
Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Hyperthermia, Induced/methods , Methacrylates/chemistry , Neoplasms/metabolism , Algorithms , Animals , Chemistry, Physical , Drug Delivery Systems , Female , Image Processing, Computer-Assisted , Magnetic Resonance Imaging , Mice , Mice, Nude , Microscopy, Fluorescence , Nanoparticles , Particle SizeABSTRACT
In most drug delivery systems the clinician does not have control over the location of drug delivery after the therapeutic has been administered. As the location of the tumor mass is often known in many patients, a therapy system which enables the clinician to play an active role in nanomedicine localization would provide an advantage. Here, we show a new approach wherein a laser can be used to tag tumor tissue and enhance the delivery of targeted polymer therapeutics. Plasmonic gold nanorods are delivered to the cancerous tissue and heated by a laser to promote a targetable, hyperthermic response. Concurrent administration of a heat shock targeted polymer therapeutic thereby enhances site specific delivery.
ABSTRACT
The field of polymer therapeutics has evolved over the past decade and has resulted in the development of polymer-drug conjugates with a wide variety of architectures and chemical properties. Whereas traditional non-degradable polymeric carriers such as poly(ethylene glycol) (PEG) and N-(2-hydroxypropyl methacrylamide) (HPMA) copolymers have been translated to use in the clinic, functionalized polymer-drug conjugates are increasingly being utilized to obtain biodegradable, stimuli-sensitive, and targeted systems in an attempt to further enhance localized drug delivery and ease of elimination. In addition, the study of conjugates bearing both therapeutic and diagnostic agents has resulted in multifunctional carriers with the potential to both "see and treat" patients. In this paper, the rational design of polymer-drug conjugates will be discussed followed by a review of different classes of conjugates currently under investigation. The design and chemistry used for the synthesis of various conjugates will be presented with additional comments on their potential applications and current developmental status.
ABSTRACT
Polymeric micelles carrying the heat shock protein 90 inhibitor tanespimycin (17-N-allylamino-17-demethoxygeldanamycin) were synthesized using poly(styrene-co-maleic acid) (SMA) copolymers and evaluated in vitro and in vivo. SMA-tanespimycin micelles were prepared with a loading efficiency of 93%. The micelles incorporated 25.6% tanespimycin by weight, exhibited a mean diameter of 74 ± 7 nm by dynamic light scattering and a zeta potential of -35 ± 3 mV. Tanespimycin was released from the micelles in a controlled manner in vitro, with 62% released in 24h from a pH 7.4 buffer containing bovine serum albumin. The micellar drug delivery systems for tanespimycin showed potent activity against DU145 human prostate cancer cells, with an IC(50) of 230 nM. They further exhibited potent anti-cancer activity in vivo in nu/nu mice bearing subcutaneous DU145 human prostate cancer tumor xenografts, with significantly higher anticancer efficacy as measured by tumor regression when compared to free tanespimycin at an equivalent single dose of 10mg/kg. These data suggest further investigation of SMA-tanespimycin as a promising agent in the treatment of prostate cancer.
Subject(s)
Antineoplastic Agents/administration & dosage , Benzoquinones/administration & dosage , Drug Carriers , Lactams, Macrocyclic/administration & dosage , Maleates/chemical synthesis , Nanoparticles , Polystyrenes/chemical synthesis , Prostatic Neoplasms/drug therapy , Animals , Antineoplastic Agents/chemistry , Benzoquinones/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Chemistry, Pharmaceutical , Dose-Response Relationship, Drug , Drug Compounding , Humans , Hydrogen-Ion Concentration , Inhibitory Concentration 50 , Injections, Subcutaneous , Kinetics , Lactams, Macrocyclic/chemistry , Light , Male , Mice , Mice, Nude , Micelles , Nanotechnology , Prostatic Neoplasms/pathology , Scattering, Radiation , Solubility , Technology, Pharmaceutical/methods , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer-docetaxel-RGDfK conjugate was synthesized, characterized, and evaluated in vitro and in vivo in comparison with untargeted low and high molecular weight HPMA copolymer-docetaxel conjugates. The targeted conjugate was designed to have a hydrodynamic diameter below renal threshold to allow elimination post treatment. All conjugates demonstrated the ability to inhibit the growth of DU145 and PC3 human prostate cancer cells and the HUVEC at low nanomolar concentrations. The targeted conjugate showed active binding to α(v)ß(3) integrins in both HUVEC and DU145 cells, whereas the untargeted conjugate demonstrated no evidence of specific binding. Efficacy at two concentrations (20 mg/kg and 40 mg/kg) was evaluated in nu/nu mice bearing DU145 tumor xenografts treated with a single dose of conjugates and compared with controls. RGDfK targeted and high molecular weight nontargeted conjugates exhibited the highest antitumor efficacy as evaluated by tumor regression. These results demonstrate that α(v)ß(3) integrin targeted polymeric conjugates with improved water solubility, reduced toxicity and ease of elimination post treatment in vivo are promising candidates for prostate cancer therapy.
Subject(s)
Acrylamides/chemistry , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Polymers/chemistry , Prostatic Neoplasms/drug therapy , Taxoids/chemistry , Taxoids/therapeutic use , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Docetaxel , Male , Mice , Mice, Nude , Taxoids/pharmacologyABSTRACT
Tumor progression is dependent on neoangiogenesis for blood supply. Neovasculature over-express α(v)ß(3) integrins which recognize the Arg-Gly-Asp (RGD) sequence in the extracellular matrix. N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing side chains terminated in cyclic RGD analogs such as RGDfK show increased accumulation in prostate tumors. Geldanamycin and their derivatives (e.g., aminohexylgeldanamycin (AH-GDM)) are benzoquinone ansamycins that have both antiangiogenic and antitumor activity. In this work the antiangiogenic and antitumor activities of targetable HPMA copolymer-RGDfK-AH-GDM conjugates were compared with non-targetable systems in vitro and in vivo. Copolymer-drug conjugates containing RGDfK in the side chains showed superior activity against endothelial and prostate cancer cell lines in vitro, as well as higher inhibition of angiogenesis in vivo. At equimolar doses of the drug, the RGDfK containing conjugates showed significantly higher antitumor activity in nude mice bearing DU-145 human prostate cancer xenografts. These findings suggest the utility of HPMA copolymer-RGDfK conjugates for targeted delivery of geldanamycin analogs with a dual mode of action.
Subject(s)
Angiogenesis Inhibitors/therapeutic use , Benzoquinones/therapeutic use , Lactams, Macrocyclic/therapeutic use , Neovascularization, Pathologic/prevention & control , Peptides, Cyclic/therapeutic use , Polymethacrylic Acids/therapeutic use , Prostatic Neoplasms/blood supply , Prostatic Neoplasms/drug therapy , Angiogenesis Inhibitors/administration & dosage , Angiogenesis Inhibitors/chemical synthesis , Angiogenesis Inhibitors/chemistry , Animals , Benzoquinones/administration & dosage , Benzoquinones/chemical synthesis , Benzoquinones/chemistry , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Endothelial Cells/drug effects , Humans , Lactams, Macrocyclic/administration & dosage , Lactams, Macrocyclic/chemical synthesis , Lactams, Macrocyclic/chemistry , Male , Mice , Mice, Nude , Molecular Structure , Molecular Weight , Neovascularization, Pathologic/pathology , Peptides, Cyclic/administration & dosage , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/chemistry , Polymethacrylic Acids/administration & dosage , Polymethacrylic Acids/chemical synthesis , Polymethacrylic Acids/chemistry , Prostatic Neoplasms/pathology , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: This study focused on the synthesis and in vitro characterization of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates for the delivery of geldanamycin to prostate cancer tumors. Conjugates were modified to incorporate WIFPWIQL peptide, which binds to cell-surface-expressed Glucose-regulated protein 78. METHODS: HPMA copolymers containing aminohexylgeldanamycin with and without WIFPWIQL peptide were synthesized and characterized, and stability in pH 7.4 and pH 5.0 buffers, complete cell culture medium, and fetal bovine serum was evaluated. The comparative cell surface expression of GRP78 in DU145 and PC3 cell lines was assessed and competitive binding to cell surface expressed GRP78 evaluated. The ability of the conjugates to inhibit cell growth was also evaluated in vitro. RESULTS: HPMA copolymer-aminohexylgeldanamycin conjugates were stable with maximal release observed in fetal bovine serum at 37°C of approximately 10% in 72 h. HPMA copolymers bearing WIFPWIQL peptide bound to cell surface expressed GRP78 with affinities comparable to free WIFPWIQL peptide and demonstrated increased cytotoxicity as compared to untargeted conjugates. CONCLUSION: HPMA copolymer aminohexylgeldanamycin conjugates bearing WIFPWIQL peptide have the ability to bind to cell-surface-expressed GRP78 and inhibit the growth of human prostate cancer cells, suggesting that the conjugates have the potential to target solid prostate cancer tumors.