Development of a high-performance liquid chromatographic–mass spectrometric assay for the specific and sensitive quantification of Ro 64-0802, an anti-influenza drug, and its pro-drug, oseltamivir, in human and animal plasma and urine

Oseltamivir phosphate (Ro 64-0796/002) is a pro-drug of the anti-influenza neuraminidase inhibitor, Ro 64-0802, and as Tamiflu™, has been developed for the treatment of both A and B strains of the disease. This paper describes an HPLC–MS–MS assay for both compounds in plasma and urine which fulfils all of the criteria for a good analytical method. It is sensitive with limits of quantification of 1 and 10 ng/ml for the pro-drug and active neuraminidase inhibitor, respectively. It is both accurate and precise with typical coefficients of variation from some 5000 quality control samples of approximately ±3 and ±6%, respectively. Extensive stability studies have demonstrated the absence of significant problems associated with the decomposition of either compound, although ex vivo hydrolysis of Ro 64-0796 to Ro 64-0802 in rodent plasma has to be prevented by the use of the esterase inhibitor, dichlorvos.     

CEA-negative glioblastoma and melanoma cells are sensitive to cytosine deaminase/5-fluorocytosine therapy directed by the carcinoembryonic antigen promoter

Recent studies have suggested that carcinoembryonic antigen (CEA)-promoter sequences are active only in CEA-positive cells, filing in the criteria for tumor specific targeting of suicide genes. However, the present study on gene therapy of colon cancer and cell-specificity of CEA promoter, provide evidence that CEA-positive and CEA-negative cells transfected with E. coli cytosine deaminase (CD) gene under the control of CEA promotor sequence are sensitive to enzyme/pro-drug therapy with 5-fluorocytosine (5-FC). Individual clones derived from the CEA-negative cell lines: melanoma Hs294T and glioblastoma T98G after transfection with CD differed profoundly in their sensitivity to 5-FC. The IC50 values for several clones of the CEA-negative cells were almost the same as for CEA-positive colon cancer cells. Such 5-FC-sensitive clones derived from the population of CEA-negative cells, present even in small number, because of the very effective bystender effect of this enzyme/pro-drug system can cause severe problems during therapy by efficiently killing surrounding normal cells. Safety is the major issue in gene therapy. Our data suggest that the safety of gene-directed enzyme pro-drug therapy (GDEPT) with CEA promoter driven expression of therapeutic genes is not so obvious as it has originally been claimed.     

Design of Helicobacter pylori glutamate racemase inhibitors as selective antibacterial agents: a novel pro-drug approach to increase exposure

High-throughput screening uncovered a pyrazolopyrimidinedione hit as a selective, low micromolar inhibitor of Helicobacter pylori glutamate racemase (MurI). Variation of the substituents around the scaffold led to low nanomolar inhibitors and improved antibacterial activity. The challenge in this program was to translate excellent enzyme inhibition into potent antibacterial activity and pharmacokinetics suitable for oral therapy. Compounds were profiled for MurI inhibition, activity against H. pylori, microsomal stability, and pharmacokinetics in mice. Iterative cycles of analog synthesis and biological testing led to compounds with substituents optimized for both low MICs (2 microg/ml) and good microsomal stability. In order to achieve high bioavailability, a novel pro-drug approach was implemented wherein a solubilizing sulfoxide moiety is oxidized in vivo to a sulfone.     

Down-regulation of PPARgamma2-induced adipogenesis by PEGylated conjugated linoleic acid as the pro-drug: Attenuation of lipid accumulation and reduction of apoptosis

This study is designed to evaluate whether the PEGylated conjugated linoleic acid (PCLA) as the pro-drug can have favorable stability, bioavailability, and anti-adipogenic activity in 3T3-L1 cells for anti-obesity when compared with conjugated linoleic acid (CLA) itself. The CLA was simply coupled to poly(ethylene glycol) (PEG) at the melting state without solvents or catalysts through ester linkages between the carboxylic group of CLA and the hydroxyl group of PEG. To confirm of PCLA as the pro-drug, CLA release from PCLA was investigated by using high-performance liquid chromatographic (HPLC), showing that CLA release from PCLA was almost 90% in a nearly continuous fashion over the next 75h. Apoptosis was promoted by both CLA- and PCLA-treatments with increasing concentrations. However, the level of cell apoptosis induced by PCLA was lower than that induced by CLA owing to the biocompatible and hydrophilic properties of PEG. Moreover, the PCLA decreased glycerol-3-phosphate dehydrogenase (GPDH) activity in 3T3-L1 cells by acting upon major adipocyte marker proteins such as PPARgamma2, C/EBPalpha, and aP2 modulators. Furthermore, either CLA or PCLA stimulated basal, but not isoproterenol-sensitive, lipolysis in our cell model, suggesting that both CLA and PCLA may stimulate lipolysis via hormone sensitive lipase (HSL)-independent mechanisms. These results suggest that the PCLA may prove to be a stable pro-drug to control the deposition of fat in the human body, and that the anti-adipogenic effect of the PCLA on 3T3-L1 cells will offer a challenging approach for anti-obesity.     

Design,synthesis and evaluation of a novel double pro-drug: INX-08189. A new clinical candidate for hepatitis C virus

We herein report a novel double pro-drug approach applied to the anti-HCV agent 2′-β-C-methyl guanosine. A phosphoramidate ProTide motif and a 6-O-methoxy base pro-drug moiety are combined to generate lipophilic prodrugs of the monophosphate of the guanine nucleoside. Modification of the ester and amino acid moieties lead to a compound INX-08189 that exhibits 10 nM potency in the HCV genotype 1b subgenomic replicon, thus being 500 times more potent than the parent nucleoside. The potency of the lead compound INX-08189 was shown to be consistent with intracellular 2′-C-methyl guanosine triphosphate levels in primary human hepatocytes. The separated diastereomers of INX-08189 were shown to have similar activity in the replicon assay and were also shown to be similar substrates for enzyme processing. INX-08189 has completed investigational new drug enabling studies and has been progressed into human clinical trials for the treatment of chronic HCV infection.     

Dual pro-drugs of 2'-C-methyl guanosine monophosphate as potent and selective inhibitors of hepatitis C virus

We have previously reported the power of combining a 5'-phosphoramidate ProTide, phosphate pro-drug, motif with a 6-methoxy purine pro-drug entity to generate highly potent anti-HCV agents, leading to agents in clinical trial. We herein extend this work with the disclosure that a variety of alternative 6-substituents are tolerated. Several compounds exceed the potency of the prior 6-methoxy leads, and in almost every case the ProTide is several orders of magnitude more potent than the parent nucleoside. We also demonstrate that these agents act as pro-drugs of 2'-C-methyl guanosine monophosphate. We have also reported the novel use of hepatocyte cell lysate as an ex vivo model for ProTide metabolism.     

Human plasma-mediated hypoxic activation of indolequinone-based naloxone pro-drugs

Hypoxia is known to occur in tissues in response to narcotic analgesic therapy using as a result of respiratory depression. The aim of this study was to synthesize a narcotic antagonist pro-drug that can be activated by tissue hypoxia to prevent the damage associated with respiratory depression. We synthesized three different pro-drugs of the narcotic antagonist naloxone utilizing indolequinone as the hypoxia-sensitive moiety. The indolequinone structure in the pro-drugs was designed to have an open reactive point at the N-1 position offering the possibility of further conjugation with macromolecules to modify the bio-availability of these pro-drugs in vivo. A pro-drug (labeled 1) where naloxone and the indolequinone moiety were linked through a carbonate bond was rapidly hydrolyzed in phosphate buffered saline. However, two additional pro-drugs (labeled 2 and 3) having carbamate linkers were stable in phosphate buffered saline for 24 h. The reductive release of naloxone from the pro-drugs was achieved in the presence of the bio-reductive enzyme DT-Diaphorase, with about 80% release occurring from the two pro-drugs in 24 h. More than 99% of naloxone was released from pro-drug 2 in 30% human plasma, however the release only occurred under hypoxic conditions. This system provides a potential means for feedback control to counter critical respiratory depression induced by narcotic analgesics.     

Insights into the redox cycle of human quinone reductase 2

NRH:quinone oxidoreductase 2 (QR2) is a cytosolic enzyme that catalyzes the reduction of quinones, such as menadione and co-enzymes Q. With the aim of understanding better the mechanisms of action of QR2, we approached this enzyme catalysis via electron paramagnetic resonance (EPR) measurements of the by-products of the QR2 redox cycle. The variation in the production of oxidative species such as H(2)O(2), and subsequent hydroxyl radical generation, was measured during the course of QR2 activity under aerobic conditions and using pure human enzyme. The effects on the activity of the following were compared: (i) synthetic (N-benzyldihydronicotinamide, BNAH) or natural (nicotinamide riboside, NRH) co-substrates; (ii) synthetic (menadione) or natural (co-enzyme Q0, Q2) substrates; (iii) QR2 modulators and inhibitors (melatonin, resveratrol and S29434); (iv) a pro-drug activated via a redox cycle [CB1954, 5-(aziridin-1-yl)-2,4-dinitrobenzamide]. The results were also compared with those obtained with human QR1. The production of hydroxyl radicals is: (i) observed whatever the substrate/co-substrate used; ii) quenched by adding catalase; (iii) not observed with the specific QR2 inhibitor S29434; (iv) observed with the pro-drug CB1954. While QR2 produced free radicals with this pro-drug, QR1 gave no EPR signal showing the strong reducing capacity of QR2. In conclusion, EPR analysis of QR2 enzyme activity through free radical production enables modulators and effective inhibitors to be distinguished.     

Exploration of in vitro pro-drug activation and futile cycling by glutathione S-transferases: thiol ester hydrolysis and inhibitor maturation

In addition to glutathione (GSH) conjugating activity, glutathione S-transferases (GSTs) catalyze "reverse" reactions, such as the hydrolysis of GSH thiol esters. Reverse reactions are of interest as potential tumor-directed pro-drug activation strategies and as mechanisms for tissue redistribution of carboxylate-containing drugs. However, the mechanism and specificity of GST-mediated GSH thiol ester hydrolysis are uncharacterized. Here, the GSH thiol esters of ethacrynic acid (E-SG) and several nonsteroidal antiinflammatory agents have been tested as substrates with human GSTs. The catalytic hydrolysis of these thiol esters appears to be a general property of GSTs. The hydrolysis of the thiol ester of E-SG was studied further with GSTA1-1 and GSTP1-1, as a model pro-drug with several possible fates for the hydrolysis products: competitive inhibition, covalent enzyme adduction, and sequential metabolism. In contrast to hydrolysis rates, significant isoform-dependent differences in the subsequent fate of the products ethacrynic acid and GSH were observed. At low [E-SG], only the GSTP1-1 efficiently catalyzed sequential metabolism, via a dissociative mechanism.     

Evaluating Baculovirus as a Vector for Human Prostate Cancer Gene Therapy

Gene therapy represents an attractive strategy for the non-invasive treatment of prostate cancer, where current clinical interventions show limited efficacy. Here, we evaluate the use of the insect virus, baculovirus (BV), as a novel vector for human prostate cancer gene therapy. Since prostate tumours represent a heterogeneous environment, a therapeutic approach that achieves long-term regression must be capable of targeting multiple transformed cell populations. Furthermore, discrimination in the targeting of malignant compared to non-malignant cells would have value in minimising side effects. We employed a number of prostate cancer models to analyse the potential for BV to achieve these goals. In vitro, both traditional prostate cell lines as well as primary epithelial or stromal cells derived from patient prostate biopsies, in two- or three-dimensional cultures, were used. We also evaluated BV in vivo in murine prostate cancer xenograft models. BV was capable of preferentially transducing invasive malignant prostate cancer cell lines compared to early stage cancers and non-malignant samples, a restriction that was not a function of nuclear import. Of more clinical relevance, primary patient-derived prostate cancer cells were also efficiently transduced by BV, with robust rates observed in epithelial cells of basal phenotype, which expressed BV-encoded transgenes faster than epithelial cells of a more differentiated, luminal phenotype. Maximum transduction capacity was observed in stromal cells. BV was able to penetrate through three-dimensional structures, including in vitro spheroids and in vivo orthotopic xenografts. BV vectors containing a nitroreductase transgene in a gene-directed enzyme pro-drug therapy approach were capable of efficiently killing malignant prostate targets following administration of the pro-drug, CB1954. Thus, BV is capable of transducing a large proportion of prostate cell types within a heterogeneous 3-D prostate tumour, can facilitate cell death using a pro-drug approach, and shows promise as a vector for the treatment of prostate cancer.     

Insights into the redox cycle of human quinone reductase 2

Abstract

NRH:quinone oxidoreductase 2 (QR2) is a cytosolic enzyme that catalyzes the reduction of quinones, such as menadione and co-enzymes Q. With the aim of understanding better the mechanisms of action of QR2, we approached this enzyme catalysis via electron paramagnetic resonance (EPR) measurements of the by-products of the QR2 redox cycle. The variation in the production of oxidative species such as H₂O₂, and subsequent hydroxyl radical generation, was measured during the course of QR2 activity under aerobic conditions and using pure human enzyme. The effects on the activity of the following were compared: (i) synthetic (N-benzyldihydronicotinamide, BNAH) or natural (nicotinamide riboside, NRH) co-substrates; (ii) synthetic (menadione) or natural (co-enzyme Q0, Q2) substrates; (iii) QR2 modulators and inhibitors (melatonin, resveratrol and S29434); (iv) a pro-drug activated via a redox cycle [CB1954, 5-(aziridin-1-yl)-2,4-dinitrobenzamide]. The results were also compared with those obtained with human QR1. The production of hydroxyl radicals is: (i) observed whatever the substrate/co-substrate used; ii) quenched by adding catalase; (iii) not observed with the specific QR2 inhibitor S29434; (iv) observed with the pro-drug CB1954. While QR2 produced free radicals with this pro-drug, QR1 gave no EPR signal showing the strong reducing capacity of QR2. In conclusion, EPR analysis of QR2 enzyme activity through free radical production enables modulators and effective inhibitors to be distinguished.     

Species-Specific Antimonial Sensitivity in Leishmania Is Driven by Post-Transcriptional Regulation of AQP1

Leishmania is a digenetic protozoan parasite causing leishmaniasis in humans. The different clinical forms of leishmaniasis are caused by more than twenty species of Leishmania that are transmitted by nearly thirty species of phlebotomine sand flies. Pentavalent antimonials (such as Pentostam or Glucantime) are the first line drugs for treating leishmaniasis. Recent studies suggest that pentavalent antimony (Sb(V)) acts as a pro-drug, which is converted to the more active trivalent form (Sb(III)). However, sensitivity to trivalent antimony varies among different Leishmania species. In general, Leishmania species causing cutaneous leishmaniasis (CL) are more sensitive to Sb(III) than the species responsible for visceral leishmaniasis (VL). Leishmania aquaglyceroporin (AQP1) facilitates the adventitious passage of antimonite down a concentration gradient. In this study, we show that Leishmania species causing CL accumulate more antimonite, and therefore exhibit higher sensitivity to antimonials, than the species responsible for VL. This species-specific differential sensitivity to antimonite is directly proportional to the expression levels of AQP1 mRNA. We show that the stability of AQP1 mRNA in different Leishmania species is regulated by their respective 3’-untranslated regions. The differential regulation of AQP1 mRNA explains the distinct antimonial sensitivity of each species.     

6-Thioguanine alters the structure and stability of duplex DNA and inhibits quadruplex DNA formation.

The ability to chemically synthesize biomolecules has opened up the opportunity to observe changes in structure and activity that occur upon single atom substitution. In favorable cases this can provide information about the roles of individual atoms. The substitution of 6-thioguanine (6SG) for guanine is a potentially very useful single atom substitution as 6SG has optical, photocrosslinking, metal ion binding and other properties of potential utility. In addition, 6-mercaptopurine is a clinically important pro-drug that is activated by conversion into 6SG by cells. The results presented here indicate that the presence of 6SG blocks the formation of quadruplex DNA. The presence of 6SG alters the structure and lowers the thermal stability of duplex DNA, but duplex DNA can be formed in the presence of 6SG. These results indicate that some of the cytotoxic activity of 6SG may be due to disruption of the quadruplex structures formed by telomere and other DNAs. This additional mode of action is consistent with the delayed onset of cytotoxicity.     

Increased recovery of brain acetylcholinesterase activity in dichlorvos-intoxicated European eels Anguilla anguilla by bath treatment with N-acetylcysteine

Organophosphate (OP) pesticides are widely used as antiparasitic chemicals in finfish aquaculture. However, current antidotes cannot be applied to treat intoxicated fish. We showed in previous studies the importance of glutathione (GSH) metabolism in pesticide resistance of the European eel Anguilla anguilla L. The present work studied the effects of the antioxidant and glutathione pro-drug N-acetyl-L-cysteine (NAC) on the recovery of European eels exposed for 96 h to a sublethal concentration (0.17 mg l(-1); 20% of its 96 h LC50) of the OP pesticide dichlorvos (2,2-dichlorovinyl dimethyl phosphate; DDVP). This insecticide and acaricide decreased muscular GSH content and increased oxidised glutathione (GSSG), lowering the GSH:GSSG ratio, which is indicative of a condition of oxidative stress. Acetylcholinesterase (AChE) and glutathione reductase (GR) activities in the brain, which were biomarkers of neurotoxicity and oxidative stress, respectively, were also highly inhibited. Recovery in a 0.5 mM (81.6 mg l(-1)) NAC concentration ameliorated muscular GSH depletion, GSH:GSSG ratio, and the inhibition of brain AChE and GR activities. Hence, this is the first evidence of improved recovery of organophosphate-poisoned fish by bath treatments.     

Calpain inhibitors and antioxidants act synergistically to prevent cell necrosis: effects of the novel dual inhibitors (cysteine protease inhibitor and antioxidant) BN 82204 and its pro-drug BN 82270

Cell death is a common feature observed in neurodegenerative disorders, and is often associated with calpain activation and overproduction of reactive oxygen species (ROS). This study investigated the use of calpain inhibitors and antioxidants in combination to protect cells against necrosis. Maitotoxin (MTX), which induces a massive influx of calcium, was used to provoke neuronal cell death. This toxin increased, in a concentration-dependent manner, both calpain activity and ROS formation. Calpain inhibitors or antioxidants inhibited MTX-induced necrosis only marginally (below 20%), whereas their association protected against cell death by 40-66% in a synergistic manner. BN 82204, which possesses both calpain-cathepsin L inhibitory and antioxidant properties, and its acetylated pro-drug BN 82270, totally protected cells at 100 microm. The pro-drug BN 82270, which had better cell penetration, was twice as effective as the active principle BN 82204 in protecting glioma C6 or neuroblastoma SHSY5Y cells against death. These results suggest the potential therapeutic relevance of using a single molecule with multiple activities (cysteine protease inhibitor/antioxidant), and warrant further in vivo investigations in models of neuronal disorders.     

Inhibitory actions of desacetylation products of phenacetin and paracetamol on prostaglandin synthetases in neuronal and glial cell lines and rat renal medulla

The inhibitory actions of phenacetin and paracetamol and of their desacetylation products on prostaglandin synthesis were studied on enzyme preparations originating from a neuronal cell line (mouse neuroblastoma, clone N2A), a glial cell line (rat astrocytoma, clone C6) and rat renal medulla. All compounds tested inhibited cultured cell and kidney prostaglandin synthetases to similar extents. p-Phenetidin and p-aminophenol, the desacetylated metabolites of phenacetin and paracetamol, were either 7–10 times or 4 times more potent than paracetamol. Thus, p-phenetidin inhibited prostaglandin synthesis as efficaciously as did acetylsalicylic acid. The possible roles of p-phenetidin as the active metabolite of phenacetin for cyclo-oxygenase inhibition in brain and of phenacetin as an organ pro-drug are discussed.     

Synthesis and evaluation of the 2,4-diaminoquinazoline series as anti-tubercular agents

The 2,4-diaminoquinazoline class of compounds has previously been identified as an effective inhibitor of Mycobacterium tuberculosis growth. We conducted an extensive evaluation of the series for its potential as a lead candidate for tuberculosis drug discovery. Three segments of the representative molecule N-(4-fluorobenzyl)-2-(piperidin-1-yl)quinazolin-4-amine were examined systematically to explore structure–activity relationships influencing potency. We determined that the benzylic amine at the 4-position, the piperidine at 2-position and the N-1 (but not N-3) are key activity determinants. The 3-deaza analog retained similar activity to the parent molecule. Biological activity was not dependent on iron or carbon source availability. We demonstrated through pharmacokinetic studies in rats that good in vivo compound exposure is achievable. A representative compound demonstrated bactericidal activity against both replicating and non-replicating M. tuberculosis. We isolated and sequenced M. tuberculosis mutants resistant to this compound and observed mutations in Rv3161c, a gene predicted to encode a dioxygenase, suggesting that the compound may act as a pro-drug.     

Danshen diversity defeating dementia

Salvia miltiorrhiza (danshen) is widely used for the clinical treatment of cerebral ischemia and cardiovascular diseases. Its diverse molecular makeup of simple and poly hydroxycinnamic acids and diterpenoid quinones are also associated with its beneficial health effects such as improved cognitive deficits in mice, protection of neuronal cells, prevention of amyloid fibril formation and preformed amyloid fibril disaggregation related to Alzheimer’s disease. Whilst the in vitro studies have therapeutic promise, the anti-dementia effect/impact of danshen however depends on its absorbed constituents and pharmacokinetic properties. Both the water and lipid danshen fractions have been shown to have low oral bioavailability and at physiological pH, the polyphenolic carboxylate anions are not brain permeable. To tap into the many neuroprotective and other biological benefits of danshen, the key challenge resides in developing danshen nanopharmaceuticals, semi-synthetic pro-drug forms of its constituents to improve its biocompatability, that is, absorption, circulation in bloodstream and optimization of BBB permeability.