ABSTRACT
Insulin resistance (IR) contributes to the pathophysiology of diabetes, dementia, viral infection, and cardiovascular disease. Drug repurposing (DR) may identify treatments for IR; however, barriers include uncertainty whether in vitro transcriptomic assays yield quantitative pharmacological data, or how to optimise assay design to best reflect in vivo human disease. We developed a clinical-based human tissue IR signature by combining lifestyle-mediated treatment responses (>500 human adipose and muscle biopsies) with biomarkers of disease status (fasting IR from >1200 biopsies). The assay identified a chemically diverse set of >130 positively acting compounds, highly enriched in true positives, that targeted 73 proteins regulating IR pathways. Our multi-gene RNA assay score reflected the quantitative pharmacological properties of a set of epidermal growth factor receptor-related tyrosine kinase inhibitors, providing insight into drug target specificity; an observation supported by deep learning-based genome-wide predicted pharmacology. Several drugs identified are suitable for evaluation in patients, particularly those with either acute or severe chronic IR.
Developing a new drug that is both safe and effective is a complex and expensive endeavor. An alternative approach is to 'repurpose' existing, safe compounds that is, to establish if they could treat conditions others than the ones they were initially designed for. To achieve this, methods that can predict the activity of thousands of established drugs are necessary. These approaches are particularly important for conditions for which it is hard to find promising treatment. This includes, for instance, heart failure, dementia and other diseases that are linked to the activity of the hormone insulin becoming modified throughout the body, a defect called insulin resistance. Unfortunately, it is difficult to model the complex actions of insulin using cells in the lab, because they involve intricate networks of proteins, tissues and metabolites. Timmons et al. set out to develop a way to better assess whether a drug could be repurposed to treat insulin resistance. The aim was to build a biological signature of the disease in multiple human tissues, as this would help to make the findings more relevant to the clinic. This involved examining which genes were switched on or off in thousands of tissue samples from patients with different degrees of insulin resistance. Importantly, some of the patients had their condition reversed through lifestyle changes, while others did not respond well to treatment. These 'non-responders' provided crucial new clues to screen for active drugs. Carefully piecing the data together revealed the molecules and pathways most related to the severity of insulin resistance. Cross-referencing these results with the way existing drugs act on gene activity, highlighted 138 compounds that directly bind 73 proteins responsible for regulating insulin resistance pathways. Some of the drugs identified are suitable for short-term clinical studies, and it may even be possible to rank similar compounds based on their chemical activity. Beyond giving a glimpse into the complex molecular mechanisms of insulin resistance in humans, Timmons et al. provide a fresh approach to how drugs could be repurposed, which could be adapted to other conditions.
Subject(s)
Drug Repositioning , Metabolic Diseases/drug therapy , Adipose Tissue/metabolism , Biomarkers/metabolism , Humans , Insulin Resistance , Metabolic Diseases/genetics , Muscles/metabolism , TranscriptomeABSTRACT
Learned associations between effects of abused drugs and the drug administration environment are important in drug addiction. Histochemical and electrophysiological studies suggest that these associations are encoded in sparsely distributed nucleus accumbens neurons that are selectively activated by drugs and drug-associated cues. Although correlations have been observed between nucleus accumbens neuronal activity and responsivity to drugs and drug cues, no technique exists for selectively manipulating these activated neurons and establishing their causal role in behavioral effects of drugs and drug cues. Here we describe a new approach, which we term the 'Daun02 inactivation method', that selectively inactivates a minority of neurons previously activated by cocaine in an environment repeatedly paired with cocaine to demonstrate a causal role for these activated neurons in context-specific cocaine-induced psychomotor sensitization in rats. This method provides a new tool for studying the causal roles of selectively activated neurons in behavioral effects of drugs and drug cues and in other learned behaviors.
Subject(s)
Cocaine-Related Disorders/physiopathology , Cocaine/pharmacology , Dopamine Uptake Inhibitors/pharmacology , Neurons/drug effects , Nucleus Accumbens/drug effects , Animals , Behavior, Animal/drug effects , Behavior, Animal/physiology , Cues , Daunorubicin/toxicity , Disease Models, Animal , Learning/drug effects , Learning/physiology , Male , Neuronal Plasticity/drug effects , Neuronal Plasticity/physiology , Neurons/physiology , Neuropharmacology/methods , Neurotoxins/toxicity , Nucleus Accumbens/physiology , Psychomotor Performance/drug effects , Psychomotor Performance/physiology , Rats , Rats, Sprague-Dawley , Rats, TransgenicABSTRACT
Poly-L-glutamic acid (PGA) has previously been demonstrated to be an effective backbone for creating a hydrophilic prodrug of the established anti-tumor agent, paclitaxel, the active agent in Taxol; this approach has obviated the need for the toxic Cremophor excipient, used to enhance the solubility of paclitaxel in the clinical formulation. In order to form hydrophilic prodrugs of the hydrophobic pro-apoptotic sphingolipid, N,N-dimethylsphingosine (DMSP), PGA was condensed with DMSP, previously modified with coumarin to allow spectroscopic detection during conjugate synthesis, to yield PGA-DMSP. Conjugates with different loadings of DMSP were prepared and evaluated for in vitro cytotoxicity against two human breast adenocarcinoma cell lines. Time- and loading-dependent expression of cytotoxicity was observed, such that endpoints essentially equivalent to those observed with free-DMSP were achieved, but in a more protracted manner, consistent with prodrug behavior. PGA-DMSP was initially evaluated for toxicity in female nude mice, and administration of high net levels of DMSP, exceeding those achievable with free-DMSP, was well-tolerated. We propose that PGA-DMSP conjugates merit evaluation for anti-tumor efficacy in pre-clinical tumor models.
Subject(s)
Chemistry, Pharmaceutical/methods , Polyglutamic Acid/chemistry , Sphingosine/analogs & derivatives , Animals , Apoptosis , Cell Line, Tumor , Drug Design , Female , Humans , Inhibitory Concentration 50 , Mice , Mice, Nude , Models, Chemical , Prodrugs/chemistry , Spectrophotometry/methods , Sphingosine/chemistryABSTRACT
Numerous human tumor types, including ovarian cancer, display a significant expression of the CD44 family of cell surface proteoglycans. To develop tumor-targeted drugs, we have initially evaluated whether the CD44 ligand hyaluronic acid (HA) could serve as a backbone for paclitaxel (TXL) prodrugs. HA-TXL was prepared by modification of previous techniques. The in vitro cytotoxicity of HA-TXL against the CD44(+) human ovarian carcinoma cell lines SKOV-3ip and NMP-1 could be significantly blocked by preincubation with a molar excess of free HA. Female nude mice bearing intraperitoneal implants of NMP-1 cells were treated intraperitoneally with a single sub-maximum tolerated dose dose of HA-TXL or with multiple-dose regimens of paclitaxel (Taxol; Mead Johnson, Princeton, NJ) to determine the effects of these regimens on host survival and intraperitoneal tumor burden, with the latter being assessed by magnetic resonance imaging. NMP-1 xenografts were highly resistant to Taxol regimens, as host survival was only nominally improved compared to controls (T//C approximately 120), whereas single-dose HA-TXL treatment significantly improved survival in this model (T//C approximately 140; P = .004). In both NMP-1 and SKOV-3ip models, MR images of abdomens of HA-TXL-treated mice obtained shortly before controls required humane sacrifice revealed markedly reduced tumor burdens compared to control mice. This study is among the first to demonstrate that HA-based prodrugs administered locoregionally have antitumor activity in vivo.
Subject(s)
Antineoplastic Agents, Phytogenic/therapeutic use , Hyaluronan Receptors/metabolism , Hyaluronic Acid/chemistry , Ovarian Neoplasms/drug therapy , Paclitaxel/therapeutic use , Animals , Cell Proliferation/drug effects , Female , Humans , Injections, Intraperitoneal , Magnetic Resonance Imaging , Mice , Mice, Nude , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Prodrugs/administration & dosage , Tumor Burden , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Coumarin derivatives of N,N-dimethylsphingosine (DMSP) were prepared and chemically characterized. They were apparently biologically equivalent to DMSP in terms of tumor cell cytotoxicity and were used to establish the rapid mitochondrial localization of this sphingolipid in tumor cells, followed closely by its marked reduction of mitochondrial membrane potential.
Subject(s)
Coumarins/metabolism , Coumarins/pharmacology , Mitochondria/metabolism , Sphingosine/analogs & derivatives , Cell Line, Tumor , Coumarins/chemistry , Humans , Membrane Potential, Mitochondrial/drug effects , Microscopy, Fluorescence , Mitochondria/chemistry , Sphingosine/chemistry , Sphingosine/metabolism , Sphingosine/pharmacologyABSTRACT
The L49 monoclonal antibody against the p97 antigen on melanomas and carcinomas was chemically conjugated to E. coli beta-galactosidase (beta-gal), forming a largely monomeric conjugate with preserved enzymatic activity. The resulting L49-beta-gal conjugate was used to activate (N-[(4"R,S)-4"-hexyloxy-4"-(1'''-O-beta-D-galactopyranosyl)butyl]daunorubicin) (1), a derivative of daunorubicin that has low cytotoxicity and high chemical stability. Addition of the conjugate to the prodrug resulted in an increase in cytotoxicity of approximately 10(5)-fold, a level of activation that is higher than any mAb-enzyme/prodrug combination yet described. Furthermore, the released drug had an IC(50) value of approximately 10 pM, making it significantly more potent than the vast majority of clinically approved anticancer drugs. The potential of this enzyme/prodrug combination for cancer therapy is discussed.
Subject(s)
Anthracyclines/metabolism , Anthracyclines/pharmacology , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Prodrugs/metabolism , beta-Galactosidase/chemistry , beta-Galactosidase/metabolism , Animals , Cell Line, Tumor , Chromatography, High Pressure Liquid , Drug Design , Inhibitory Concentration 50 , Kinetics , Mice , Molecular Structure , Molecular Weight , Prodrugs/chemistryABSTRACT
Bis(pivaloyloxymethyl) thymidine 5-phosphate (POM(2)-dTMP) has been investigated as a membrane-permeable prodrugs of dTMP. The growth inhibitory activity of POM(2)-TMP has been compared with thymidine (TdR) in wild type CCRF CEM cells (CEM) and a strain that lacks TdR kinase (CEM tk-). After 72 h incubation at 37 degrees C, TdR showed significant antiproliferative activity (IC(50)=27 microM) against CEM cells but was weakly effective (IC(50)=730 microM) against the mutant cell line. By comparison, bis(pivaloyloxymethyl) thymidine 5'-monophosphate (POM(2)-dTMP) was equally inhibitory (IC(50)=5 microM) to both cell lines. The growth inhibitory effects were reversed by deoxycytidine. Cellular [methyl-(3)H]dTTP pools increased linearly over 2h during incubation of CEM or CEM tk- with 5 microM POM(2)-[methyl-(3)H]dTMP. The incorporation of [methyl-(3)H]TdR into HClO(4)-insoluble cell residue by CEM tk- was <0.1% that of CEM and did not increase over 1h. In contrast, CEM tk- incorporated radioactivity from POM(2)-dTMP into acid insoluble residue at a rate 59% that of CEM. These results demonstrate that POM(2)-dTMP can penetrate into cells and serve as a source of dTMP.
Subject(s)
Prodrugs/metabolism , T-Lymphocytes/metabolism , Thymidine Kinase/deficiency , Thymidine Monophosphate/metabolism , Thymidine Monophosphate/pharmacology , Carbon Radioisotopes , Cell Line, Tumor , Cell Proliferation/drug effects , Deoxycytidine/metabolism , Deoxycytidine/pharmacology , Dose-Response Relationship, Drug , Humans , Inhibitory Concentration 50 , Kinetics , Nuclear Magnetic Resonance, Biomolecular , Permeability , Thymidine/pharmacology , Thymidine Kinase/metabolismABSTRACT
PURPOSE: We previously reported the synthesis of bis(pivaloyloxymethyl) 2',3'-dideoxyuridine 5'-monophosphate (POM2-ddUMP) (1a) as a membrane-transport prodrug formulation of the free parent nucleotide, ddUMP. Although successful at delivering ddUMP into cells in culture, POM2-ddUMP was rapidly degraded by plasma carboxylate esterases after intravenous administration to experimental animals, and therefore has limited therapeutic potential as a systemically administered prodrug. We now report the synthesis of bis(N,N'-dimethylcarbamoyloxymethyl)- and bis(N-piperidinocarbamoyloxymethyl) 2',3'-dideoxyuridine 5'-monophosphate [DM2-ddUMP (1b) and DP2-ddUMP (1c), respectively], analogues of POM2-ddUMP that were designed to be more resistant to degradation by plasma esterases. METHODS: After entering cell by passive diffusion, it was anticipated that loss of one of the carbamoyloxymethyl groups of 1b and 1c would occur by spontaneous chemical hydrolysis to give the intermediate phosphodiesters, 2b and 2c. Cleavage of the remaining carbamoyloxymethyl groups by cellular phosphodiesterase I would generate ddUMP. 1b and 1c were prepared by condensation of 2',3'-dideoxyuridine (ddU) with the appropriate bis(N-alkylcarbamoyloxymethyl) phosphate in DMA in the presence of triphenylphosphine and diethyl azodicarboxylate (the Mitsunobo reagent). RESULTS: The half-lives of 1b and 1c when incubated at a concentration of 10(-4) M in human plasma at 37 degrees C were 3.5 h and 3.7 h, respectively, similar to the half-lives observed under the same temperature conditions in 0.05 M aqueous phosphate buffer, pH 7.4. By contrast, the half-life of the POM2 prodrug, 1a, in plasma was only 5 min. The initial products of degradation of 1b and 1c were the phosphodiesters 2b and 2c. The latter compounds gave rise to ddUMP when incubated with snake venom phosphodiesterase I. CONCLUSION: These findings support the premise inherent in the design of 1b and 1c, namely that the carbamate prodrugs are far more resistant to hydrolysis by plasma carboxylate esterases than their POM counterparts and can revert to the free parent 5'-mononucletides by successive chemical and enzymatic hydrolysis. Further studies of 1b and 1c as membrane-permeable prodrugs of ddUMP are in progress.
Subject(s)
Dideoxynucleosides/chemistry , Prodrugs/chemistry , Animals , Crotalid Venoms/antagonists & inhibitors , Crotalid Venoms/enzymology , Dideoxynucleosides/pharmacokinetics , Dideoxynucleotides , Esters , Humans , Prodrugs/pharmacokinetics , Uridine Monophosphate/analogs & derivativesABSTRACT
Paclitaxel (Taxol) has demonstrated clinical activity in non-small-cell lung cancer (NSCLC), but its use has not led to marked improvements in survival. This ineffectiveness can in part be attributed to inadequate delivery of effective drug levels to the lung via systemic administration and to drug resistance mechanisms. Locoregional drug administration and the use of drug copolymers are possible approaches to address these issues. In this study, we evaluated the activity of a poly(L-glutamic acid)-paclitaxel (PGA-TXL) formulation administered by intratracheal injection to mice bearing orthotopic human NSCLC tumors (H460, H358). H460 cells were found to be sensitive to paclitaxel and PGA-TXL in vitro, in a time- and concentration-dependent manner. In preliminary acute toxicity studies, PGA-TXL administered by intratracheal injection was found to be much less toxic than paclitaxel, as anticipated. Mice into which H460 cells had been implanted by intratracheal injection were given single-dose intratracheal treatments with paclitaxel (1.2 or 2.4 mg/kg) or with PGA-TXL (15 mg/kg, paclitaxel equivalents) 1 week later. When the mice were sacrificed at up to 65 days after tumor implantation, they were evaluated grossly for tumor at bronchial, neck, and lung sites. Control mice had tumors in 60% of all three sites, and all of the control mice had tumors in at least one site. The low- and high-dose Taxol groups had fewer incidences at these three sites (27-33%) and 60-80% of these mice had tumors in at least one site. The PGA-TXL mice displayed a low (13%) incidence at these sites, and only 40% had detectable tumors. In a subsequent survival study with the intratracheal H358 model, control mice had a mean life span of 95 days, whereas both the intratracheal Taxol (2.5 mg/kg, every 7th day for three doses) and the intratracheal PGA-TXL (20 mg/kg, paclitaxel equivalents, every 7th day for three doses) groups had improved survival (mean life spans: 133.5 and 136.5 days, respectively). In pilot studies intended to compare the feasibility of the development of paclitaxel aerosols suitable for clinical application, based either on Cremophor solutions or on PGA backbones, only the latter gave acceptable particle size distributions and flow rates. These results encourage the development and application of Cremophor-free copolymer formulations of paclitaxel for locoregional treatment (e.g., as aerosol) of endobronchial malignant diseases.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Agents/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Lung Neoplasms/drug therapy , Paclitaxel/pharmacology , Animals , Cell Line, Tumor , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm , Female , Humans , Immunohistochemistry , Male , Mice , Mice, Inbred ICR , Mice, Nude , Neoplasm Transplantation , Polyglutamic Acid/chemistry , Polymers , Prodrugs/pharmacology , Time FactorsABSTRACT
PURPOSE: Suicide gene therapy offers the potential to increase the selective toxicity of antitumor agents by intratumoral expression of exogenous enzymes that convert nontoxic prodrugs to toxic products. The use of herpes simplex virus thymidine kinase with ganciclovir, and E. coli cytosine deaminase with 5-fluorocytosine are well-known examples of this approach. The purpose of this study was to investigate a novel suicide gene therapy using E. coli beta-galactosidase (beta-gal) as the prodrug-activating enzyme. Advantages of this approach include: (1) the ability to use prodrugs that are cleaved by beta-gal to agents that are known to possess activity against human solid tumors, and (2) the extensive experience gained with targeting beta-gal to specific tumors in experimental animals and in humans. METHODS: Two different structural types of anthracycline prodrugs, N-[4"-(beta- D-galactopyranosyl)-3"-nitrobenzyloxycarbonyl]daunomycin (Daun02) and N-[(4" R,S)-4"-ethoxy-4"-(1"'- O-beta- D-galactopyranosyl)butyl]daunorubicin (gal-DNC4) were investigated. The prodrugs were evaluated as substrates for beta-gal. Cytotoxicity studies of Daun02 were conducted against a murine tumor (Panc02), two human breast tumors (MCF-7 and T47D), and three human prostate tumors (PC3, DU145 and LNCAP) that had been transduced to express beta-gal. Antitumor studies of Daun02 were conducted against mouse tumor Panc02 xenografts implanted subcutaneously. RESULTS: Daun02 was a good substrate for beta-gal. By comparison, gal-DCN4 was a poor substrate. Except for PC3, the beta-gal-transduced tumors showed 3- to 60-fold increased sensitivity to Daun02 compared with mock-transduced control cells. Daunomycin was formed from Daun02 in tissue culture medium containing beta-gal-transduced cell lines but was not observed in the medium from mock-transduced controls. In vivo therapeutic studies of Daun02 against the Panc02 tumor in athymic mice showed no significant inhibition of tumor growth. Pharmacokinetic studies showed limited distribution of the prodrug beyond the vascular space. CONCLUSIONS: E. coli beta-gal may be useful as a prodrug-activating enzyme in suicide gene therapy and has the potential to increase the selective toxicity of conventional antitumor agents. Although this approach worked well against tumor cells in vitro, it was not effective against a xenograft model in vivo, apparently because of poor drug-tissue distribution.
Subject(s)
Antibiotics, Antineoplastic/therapeutic use , Escherichia coli/enzymology , Genetic Therapy/methods , Neoplasms, Experimental/therapy , Prodrugs/therapeutic use , beta-Galactosidase/metabolism , Animals , Daunorubicin/therapeutic use , Female , Genetic Vectors , Humans , Male , Mice , Mice, Inbred BALB C , Neoplasms, Experimental/enzymology , Neoplasms, Experimental/pathology , Transduction, Genetic , Transfection , Tumor Cells, Cultured/drug effects , beta-Galactosidase/geneticsABSTRACT
Previous preclinical studies with ectopic tumor models have demonstrated remarkable improvements in the therapeutic profile of paclitaxel, formulated as a copolymer with poly-L-glutamic acid, compared with paclitaxel in the clinical formulation, Taxol. In this study, we evaluated these formulations in two human ovarian carcinoma xenograft models, NMP-1 and HEY, in nude mice. i.p. implantation in female nude mice of either cell line gave rise to progressive disease within the peritoneum, in the parenchyma of visceral organs, and eventually at extraperitoneal sites; the resultant, increasing morbidity then required host sacrifice. i.p. administration of multiple-dose Taxol at its maximum tolerated dose 1 week after tumor implantation afforded minimal or no increased survival compared with controls in either model. Consistent with the predictions of drug copolymer behavior, paclitaxel, as the poly-L-glutamic acid-paclitaxel copolymer, displayed much less toxicity than Taxol in these hosts. When evaluated for antitumor efficacy in both the Taxol-resistant NMP-1 and HEY models, significant improvement in survival, and even some cures, were observed after a single i.p. treatment with this copolymer. The observed antitumor response correlated with histopathological analysis of peritoneal and extraperitoneal tumor burden in comparing control HEY mice sacrificed near the onset of morbidity with mice receiving paclitaxel copolymer. We conclude that both the i.p. NMP-1 and HEY models have significant value in establishing the efficacy of candidate agents, which might address Taxol-resistant human ovarian carcinoma. Furthermore, the poly-L-glutamic acid-paclitaxel copolymer has a superior therapeutic profile in these Taxol-resistant compartmental models.